Metabolic inhibition increases glutamate susceptibility on a PC12 cell line

Metabolic Inhibition Increases GlutamateSusceptibility on a PC12 Cell LineClaudia Pereira,1 Maria S. Santos,2 and Catarina Oliveira1*1Center for Neuroscience of Coimbra, Faculty of Medicine, University of Coimbra, Coimbra, Portugal2Department of Zoology, University of Coimbra, Coimbra, Portugal

The effect of energetic metabolism compromise, ob-tained by chemical induction of hypoglycaemia (glu-cose deprivation), hypoxia (mitochondrial respiratorychain inhibition), and ischaemia (hypoglycaemia plushypoxia), on glutamate toxicity was analyzed on PC12cells. The respiratory status of these cells, measuredby the MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide] assay, was significantly de-creased after metabolic inhibition induced by isch-aemia, but it was not affected by both hypoglycaemiaand hypoxia. Under hypoglycaemia, but not underhypoxia, ATP levels were significantly reduced (from12.676 0.48 to 5.386 1.41 nmol/mg protein). How-ever, ischaemic-like conditions greatly potentiated thedecline of ATP levels (95% decrease) observed afterhypoglycaemia. The influence of metabolic inhibitionon glutamate-induced cytotoxicity was also analyzed.When the cells were preincubated under conditionsthat deplete ATP (hypoglycaemia and ischaemia), theinhibition of MTT reduction, measured after gluta-mate incubation, was potentiated. This effect could bereverted when vitamin E and idebenone were presentduring the induction of metabolic inhibition. The ATPlevels above which glutamate susceptibility was en-hanced were also determined. These results indicatethat glutamate toxicity on PC12 cells, which occurs bya mechanism independent ofN-methyl-D-aspartate(NMDA) receptor activation, can be enhanced by thedepletion of intracellular ATP upon metabolic stress;it is dependent on the extent of ATP depletion andseems to involve the generation of free radicals. It canbe concluded that under ischaemic conditions, thedeleterious effects of glutamate can be potentiated bythe energetic compromise associated with this patho-logic situation. J. Neurosci. Res. 51:360–370, 1998.r 1998 Wiley-Liss, Inc.

Key words: ischaemia; hypoglycaemia; hypoxia; mito-chondria; ATP levels; NMDA receptor

INTRODUCTIONUnder aerobic conditions, glucose is the major

substrate for brain energy metabolism. Interruption of

blood flow (ischaemia) deprives the brain of both glucose(hypoglycaemia) and oxygen (hypoxia), which rendersthe brain vulnerable to injury. One of the main biologicevents occurring immediately after tissue ischaemia is adecrease in cellular ATP levels (Raichle, 1983). If theadverse conditions persist, a cascade of biologic eventsoccurs within the cell that leads to irreversible damageand eventually to cell death. One mechanism by whichischaemic neuronal damage is mediated is the overstimu-lation of the N-methyl-D-aspartate (NMDA) glutamatereceptor subtype, caused by an extracellular accumula-tion of excitatory amino acids (EAAs), glutamate playinga predominant role (Benveniste et al., 1984). Manylaboratories have shown the beneficial effects of thepresence of NMDA antagonists by using in vivo and invitro models of focal ischaemia, hypoxia, and hypoglycae-mia (Choi and Rothman, 1990).

Evidence is accumulating that glutamate can play adeleterious role under certain adverse conditions. Anyseries of events that reduce the energy level in any subsetof neurons expressing the NMDA receptor would renderthese neurons vulnerable to the toxic effects of glutamate,creating the environment in which any other effect, suchas excitotoxicity, must operate. Disruption of ATP synthe-sis may lead to partial neuronal depolarization withactivation of voltage-dependent NMDA receptors andsecondary excitotoxic neuronal damage (Henneberry etal., 1989). However, the possibility of alternative mecha-nisms by which glutamate susceptibility is increasedunder conditions of metabolic compromise has not beenquestioned.

The aim of this work was to analyze whether, in theabsence of NMDA receptor activation, the glutamatetoxic potency increased after perturbation of the energeticmetabolism. The correlation between ATP levels and

Contract grant sponsor: Portuguese Research Council; Contract grantsponsor: Human Capital Mobility Program; Contract grant number:ERB 4050 PL 932039.

*Correspondence to: Catarina Resende de Oliveira, Center for Neuro-science of Coimbra, Faculty of Medicine, University of Coimbra, 3000Coimbra, Portugal. E-mail: [email protected]

Received 1 August 1997; Accepted 1 September 1997

Journal of Neuroscience Research 51:360–370 (1998)

r 1998 Wiley-Liss, Inc.

increased glutamate toxicity after metabolic inhibitionalso was studied. For this purpose we analyzed the effectof metabolic inhibition, by inducing hypoglycaemic (glu-cose deprivation), hypoxic (mitochondrial respiratoryinhibition), and ischaemic-like conditions (hypoxia plushypoglycemia), on the reduction of the tetrazolium saltMTT by the mitochondria and on intracellular ATP levels.We also determined the influence of ATP depletion onglutamate cytotoxicity in a PC12 cell line. Finally, theeffect of the antioxidants vitamin E and idebenone wasanalyzed to determine whether the increased productionof reactive oxygen species is involved on PC12 cellssusceptibility to glutamate upon metabolic stress. Theresults presented here indicate that glutamate toxicity,which occurs independently of NMDA receptor activa-tion, can be enhanced under conditions that decreaseintracellular ATP levels and that the increased glutamatesusceptibility of PC12 cells is dependent on the extent ofATP depletion and free radicals production.

MATERIALS AND METHODSCell Culture

Stock cultures of PC12 cells (Greene and Tischler,1976), purchased from American Type Culture Collection(ATCC), were grown routinely in 75-cm2 tissue cultureflasks in RPMI 1640 (Sigma, St. Louis, MO) supple-mented with 10% heat-inactivated horse serum (GIBCORBL, UK) and 5% heat-inactivated fetal calf serum(Biochrom KG, Germany), to which penicillin (50 U/ml)and streptomycin (50 µg/ml) were added, and maintainedat 37°C in a humidified incubator containing 95% air and5% CO2. The cells were passed twice a week. Cells wereplated at 50,000 cells/cm2 on poly-L-lysine-coated (10µg/ml) 24-well plates (0.5 ml/well), for toxicity experi-ments, and on 12-well plates (1 ml/well) for ATPmeasurements.

Toxicity StudiesTo evaluate the dose-response and time-course

glutamate-induced toxicity, 24–48 hr after seeding, themedium was renewed by fresh culture medium contain-ing the desired concentration of glutamate. The involve-ment of the NMDA receptor on glutamate toxicity wasdetermined after incubation of PC12 cells for 3 hr with100 or 500 µM glutamate in Krebs salt solution (140 mMNaCl, 5 mM KCl, 1.5 mM CaCl2, 5.6 mM glucose, 1 mMNaH2PO4, 20 mM HEPES-NaOH, pH 7.4), in the pres-ence or in the absence of Mg21, and in the presence of 10µM glycine. The effect of 5 µM MK-801, an antagonist ofthe NMDA glutamate receptor subtype, was tested on thetoxicity of 500 µM glutamate, in the absence of Mg21 andin the presence of 10 µM glycine. The toxic effect of 100µM glutamate was analyzed, immediately after a 30-min

incubation or 24 hr after this short incubation period,under several conditions: in the presence of both glucoseand Mg21; in the presence of glucose and the absence ofMg21; in the presence of Mg21 and the absence ofglucose and in the absence of both glucose and Mg21. Theeffect of hypoglycaemic (glucose deprivation), hypoxic(mitochondrial respiratory chain inhibition), or ischaemic-like conditions (hypoglycaemia plus hypoxia) on PC12cells mitochondrial function, ATP levels and glutamate-induced cell damage were evaluated as follows: 24–48 hrafter seeding, PC12 cells were incubated for 30 min inglucose (5.6 mM) or glucose free Krebs salt solution (140mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 1mM NaH2PO4, 20 mM HEPES-NaOH, pH 7.4) in thepresence or in the absence of the mitochondrial respira-tory chain inhibitors (Sigma) rotenone (20 µM), amytal (2mM), antimycin A (1 µg/ml), KCN (5 mM), or oligomy-cin (5 µg/ml). In some experiments, the preincubation inglucose-free medium for 30 min, in the presence or in theabsence of KCN, or during 5, 10, 15, and 20 min inglucose-free medium, was followed by an additionalincubation for 3 hr in the presence of glutamate. Whenantioxidants were tested, cells were preincubated for 24hr in the presence of 10 µM vitamin E (a-tocopherol-succinate, Sigma) or 1 µM idebenone (generous gift fromSeber, Portugal) and then exposed to hypoglycaemic orischaemic conditions for 30 min, also in the presence ofthe antioxidants. Finally, cells were washed and incu-bated for an additional 3 hr in the absence or in thepresence of glutamate. Media containing rotenone, anti-mycin A, oligomycin, vitamin E, or idebenone were madeby a 1,000-fold dilution of concentrated solutions pre-pared in 100% ethanol (0.1% ethanol had no protective ortoxic effect by itself). Control cultures were conducted inthe absence of chemical agents but in the presence of0.1% ethanol. After the desired incubation period, theculture medium and/or the cells were analyzed for MTTreduction (Mosmann, 1983) or lactate dehydrogenase(LDH) content (Duval et al., 1990).

Assay for Cell InjuryThe determination of the mitochondrial function

was performed by measuring the MTT reduction abilityof PC12 cells, according to the method of Mosmann(1983). In brief, MTT was dissolved in PBS at 5 mg/mland was 10-fold diluted in serum-free RPMI 1640medium. After incubation of the cells with the com-pounds to be tested, the medium was aspirated, and 0.5 mlof MTT-containing medium was added. After an addi-tional 3-hr incubation at 37°C, 0.5 ml isopropanol/HClwas added to each well, and the absorbance at 570 nm, ofsolubilized MTT formazan products, was measured.Results were expressed as the percentage (%) of MTT

Glutamate Susceptibility After Metabolic Inhibition 361

reduction, assuming the absorbance of control cells as100%.

PC12 cell viability was assessed by monitoring theactivity of the cytoplasmic enzyme LDH in the extracellu-lar incubation medium. LDH activity was measuredspectrophotometrically according to the method of Berg-meyer and Brent (1974), by following the rate of conver-sion of reduced (NADH) to oxidized (NAD1) nicotin-amide adenine dinucleotide at 340 nm. LDH activity inthe cells was determined after incubation in a hypotonicsolution containing 15 mM Tris, at pH 7.4. LDH leakagewas expressed as the percentage (%) of the total LDHactivity (LDH in the medium1 LDH in the cells).

Analysis of Adenine NucleotidesAfter the incubation period, the medium was re-

moved, and PC12 cells were extracted, in ice, with 0.3 Mperchloric acid. The cells were scraped from the wells andcentrifuged at 15,8003 g for 5 min. The pellets weresolubilized with 1 M NaOH and analyzed for total proteincontent by the Sedmak method (Sedmak and Grossero,1977), using bovine serum albumin as standard. Thesupernatants were neutralized with 10 M KOH in 5 MTris and centrifuged at 15,8003 g for 5 min. Theresulting supernatants were assayed for adenine nucleo-tides by separation in a reverse-phase HPLC, as describedby Stocchi et al. (1985). The chromatographic apparatusused was a Beckman System Gold, consisting of a 126Binary Pump Model and a 166 Variable UV detector,controlled by a computer. The column used was aLichrospher 100 RP-18 (5 µm) from Merck (Germany).An isocratic elution with 100 mM KH2PO4 buffer, at pH7.4, and 1% methanol was performed at a flow rate of 1.2ml/min. The adenine nucleotides were detected at 254nm, for 6 min.

Data AnalysisThroughout the text, data were expressed as means6

SEM of triplicate determinations, from at least threeindependent experiments. Statistical significance analysiswas determined by using the unpaired Student’st-test oranalysis of variance (ANOVA;P value, 0.05 wasconsidered significant).

RESULTSDose- and Time-Dependent Glutamate Toxicity

The dose-dependent glutamate toxicity was evalu-ated by determining the percentage (%) of MTT reductionupon incubation of PC12 cells for 24 hr with increasingglutamate concentrations in the range 0.1–10 mM. Asshown in Figure 1A, increasing glutamate concentrationsinduced a decrease in mitochondrial function in a dose-

dependent manner, with a significant decrease of MTTreduction ability measured after treatment of the cellswith 0.5 mM glutamate (83.26 3.60% MTT reduction).The maximal response was observed for 5 mM glutamateconcentration with a percentage (%) of MTT reduction of

Fig. 1. Dose-response curve (A) and time course (B) of theeffect of glutamate on PC12 cell MTT reduction. Cells wereplated and grown for 24 hr in culture medium, and then theywere switched to fresh medium in the absence or in the presenceof glutamate. After the desired incubation period, control andglutamate-treated cells were analyzed for their ability to reducethe tetrazolium salt MTT. Data, expressed as the percentage (%)of control values, are the arithmetic mean6 SEM of triplicatedeterminations of 3–17 different experiments. ***P , 0.001;** P , 0.01; *P , 0.05, significantly different compared withcontrol conditions, in the absence of glutamate.

362 Pereira et al.

53.1 6 1.77%, similar to that measured for the highestglutamate concentration tested (51.006 2.50% MTTreduction in the presence of 10 mM glutamate). Thetime-dependent glutamate toxicity was analyzed afterincubation of the cells with 0.5 or 10 mM glutamateduring 1, 3, 6, 12, and 24 hr. Glutamate induced adecrease in mitochondrial function in a time-dependentmanner (Fig. 1B), with a slight but significant reductionin cellular redox activity after 12 hr incubation with 0.5 or10 mM glutamate, which increased upon 24-hr incu-bation.

Influence of Metabolic Inhibition on PC12 CellsRedox State and on ATP Levels

The % of MTT reduction was determined afterincubation of PC12 cells in the presence of glucose and ina glucose free-medium, in the presence or in the absenceof rotenone, amytal, antimycin A, KCN, and oligomycin(Fig. 2). When glucose was present in the medium, the %of MTT reduction was not significantly reduced byincubation with the mitochondrial respiratory chain inhibi-tors. However, when the incubation, in the presence ofthese mitochondrial respiratory chain inhibitors, wasperformed in the absence of glucose, the % of MTTreduction was significantly decreased (79.376 6.08%,48.636 15.19%, 60.406 1.48%, 49.096 5.96%, and50.486 4.76% in the presence of rotenone, amytal,antimycin A, KCN, and oligomycin, respectively). Theincubation of PC12 cells in a glucose-free medium, in theabsence of the mitochondrial respiratory chain inhibitors,did not inhibit the reduction of MTT by the mitochondrialdehydrogenases.

The intracellular levels of ATP were determined onPC12 cells after incubation for 30 min in a glucose orglucose free-medium, in the presence or in the absence ofrotenone, amytal, antimycin A, KCN, or oligomycin(Table I). In the presence of glucose, the ATP levelsdetermined after incubation with the inhibitors of themitochondrial respiratory chain (hypoxic-like conditions)were not significantly different from those determinedunder control conditions (ATP levels of 12.026 1.24,14.186 0.10, 11.466 0.04, 10.776 2.13 and 12.7561.38 nmol/mg protein were determined after incubationwith 20 µM rotenone, 2 mM amytal, 1 µg/ml antimycin A,5 mM KCN, or 5 µg/ml oligomycin, respectively, whereasin control cells ATP levels were 12.6746 0.481 nmol/mgprotein). However, when PC12 cells were incubated for30 min in a glucose free-medium (hypoglycaemic-likeconditions), a significant decrease in ATP levels wasobserved (5.386 1.41 nmol/mg protein compared with12.6746 0.481 nmol/mg protein determined in cellsincubated for 30 min in a medium with glucose). Thisdepletion of ATP, observed under hypoglycaemic condi-tions, was greatly potentiated in the presence of theinhibitors of the mitochondrial respiratory chain (isch-

aemic-like conditions) with levels of ATP of 0.636 0.04,0.556 0.05, 0.626 0.07, 1.426 0.71, and 0.766 0.26nmol/mg protein, in the presence of rotenone, amytal,antimycin A, KCN, and oligomycin, respectively.

Potentiation of Glutamate-Induced Toxicityby Metabolic Inhibition: Protective Effectof Antioxidants

The % of MTT reduction was determined after a3-hr incubation in the presence of 0.5 and 10 mMglutamate in a medium containing glucose, after the cellshave been submitted to the following treatments: 30 minin the presence of glucose (normoxic conditions); 30 minin a glucose free-medium (hypoglycaemic conditions);30 min in the presence of glucose and 5 mM KCN(hypoxic conditions) or 30 min in the absence of glucoseand in the presence of 5 mM KCN (ischaemic conditions;Fig. 3A–D). Both glutamate concentrations, 0.5 and 10mM, were not able to decrease the % of MTT reductionafter a 3-hr incubation in a medium containing glucose

Fig. 2. Effect of metabolic inhibition induced by hypoglycae-mic, hypoxic, or ischaemic-like conditions on the ability toreduce MTT by PC12 cells. Hypoglycaemia was induced in theabsence of glucose; hypoxia was induced with the mitochon-drial respiratory chain inhibitors rotenone (20 µM), amytal (2mM), antimycin A (1 µg/ml), cianide (5 mM), or oligomycin (5µg/ml), and ischaemia was induced by mitochondrial respira-tory chain inhibitors in the absence of glucose. Control cells, inthe absence of chemical agents, and treated cells were incubatedfor 30 min in Na1 medium and were then analyzed for theirability to reduce the tetrazolium salt MTT. Data are thearithmetic mean6 SEM of triplicate determinations of 3–15different experiments. ***P , 0.001; **P , 0.01; *P , 0.05,significantly different compared with control conditions.


(97.876 2.39% and 100.956 3.67% of MTT reduction,respectively; Fig. 3A). When PC12 cells were preincu-bated in the absence of glucose (Fig. 3B), a significantdecrease in the % of MTT reduction, compared withcontrol conditions, was observed in the presence of 10mM glutamate (the % of MTT reduction was 83.43261.801%) and also in the presence of 0.5 mM glutamate(the % of MTT reduction was 87.3056 2.297%). Thisdecrease in the % of MTT reduction, determined afterincubation with 0.5 or 10 mM glutamate upon hypoglycae-mic conditions, was prevented by the antioxidants vita-min E (in the presence of vitamin E, the % of MTTreduction induced by 0.5 and 10 mM glutamate was94.1636 3.374% and 96.4006 8.696%, respectively)and idebenone (in the presence of idebenone, the % ofMTT reduction induced by 0.5 and 10 mM glutamate was94.5806 2.543% and 98.4506 5.147%, respectively;Fig. 4A). If in addition to glucose deprivation, KCN waspresent during preincubation, the % of MTT reductiondetermined after glutamate incubation was potentiated(Fig. 3D): the % of MTT reduction was 68.2476 3.279%in the presence of 0.5 mM glutamate and 65.62863.219% in the presence of 10 mM glutamate. When thecells were treated, for 30 min, with KCN in the absence of

glucose and the medium was then removed and replacedby fresh medium, the mitochondrial function determined3 hr after KCN removal was higher than that determinedunder the same conditions, but in the presence ofglutamate (94.356 2.802% of MTT reduction). Theantioxidants vitamin E and idebenone also protected thecells against the glutamate-induced inhibition of MTTreduction observed after ischaemic conditions (Fig. 4B).The % of MTT reduction determined after exposure ofPC12 cells to 0.5 mM glutamate for 3 hr was 94.0363.357% and 96.7756 7.823%, respectively, when isch-aemic-like conditions were induced in the presence ofvitamin E or idebenone. Similarly, the % of MTTreduction was 98.7716 5.508% and 99.226 9.129%,respectively, when ischaemic-like conditions were in-duced in the presence of vitamin E or idebenone and thecells were then exposed to 10 mM glutamate. In thepresence of glucose, preincubation with KCN beforeexposure to 0.5 or 10 mM glutamate for 3 hr (Fig. 3C) didnot significantly affect the reduction of MTT by PC12cells (95.6286 3.239% of MTT reduction).

Relation Between Intracellular ATP Levelsand Increased Glutamate Susceptibility

The % of MTT reduction by PC12 cells wasdetermined after a 3-hr incubation in the presence of 0.5and 10 mM glutamate in a medium containing glucose,after the cells were submitted to hypoglycaemic-likeconditions in a glucose free-medium for 5, 10, 15, 20, and30 min (Fig. 5A). Preincubation of PC12 cells during 5min in glucose free-medium before incubation withglutamate for 3 hr did not affect significantly the reduc-tion of MTT. However, when the cells were preincubatedfor 10 min in the absence of glucose, a significantdecrease in the % of MTT reduction, compared withcontrol conditions, was observed in the presence of 0.5mM glutamate (the % of MTT reduction was 79.37565.963%) and also in the presence of 10 mM glutamate(the % of MTT reduction was 81.7866 4.370%). Similarresults were observed when the cells were preincubatedfor 15, 20, or 30 min in glucose-free medium beforeexposure to 0.5 or 10 mM glutamate.

ATP levels also were determined after incubation ofPC12 cells for 5, 10, 15, 20, and 30 min in the absence ofglucose (Fig. 5B). The intracellular levels of ATP werenot statistically affected after a 5-min incubation inglucose-free medium (16.2416 1.779 nmol/mg proteinin the absence of glucose in comparison with15.7326 1.808 nmol/mg protein in the presence ofglucose). However, it was observed that, after a 10-minincubation in glucose-free medium, intracellular ATPlevels decreased significantly, compared with those deter-mined after incubation in glucose medium during thesame period of time (8.8636 1.85 nmol/mg protein inglucose-free medium and 13.9036 0.623 nmol/mg pro-

TABLE I. Intracellular ATP Levels of PC12 Cells Submittedto Metabolic Inhibition (Hypoglycaemic, Hypoxic, or Ischaemic-Like Conditions)

Experimental ConditionATP Levels

(nmol/mg protein)

Control 12.676 0.48 (n5 8)Hypoglycaemia 5.386 1.41 (n5 4)**Hypoxia

Rotenone (ROT) 12.026 1.24 (n5 4)Amytal (AMY) 12.186 0.10 (n5 4)Antimycin (ANT) 11.466 0.04 (n5 4)KCN (CN) 10.776 2.13 (n5 5)Oligomycin (OL) 12.756 1.38 (n5 4)

IschaemiaHypoglycaemia plus Hypoxia (ROT) 0.636 0.04 (n5 4)***

###Hypoglycaemia plus Hypoxia (AMY) 0.556 0.05 (n5 4)***

###Hypoglycaemia plus Hypoxia (ANT) 0.626 0.07 (n5 4)***

###Hypoglycaemia plus Hypoxia (CN) 1.426 0.71 (n5 4)***

##Hypoglycaemia plus Hypoxia (OL) 0.766 0.26 (n5 4)***

##

Hypoglycaemia, hypoxia, and ischaemia were induced as described inMaterials and Methods. Control and treated cells were incubated for 30min, and then ATP was determined by reverse-phase HPLC with UVdetection. Data are the arithmetic mean6 SEM of triplicate determina-tions of the mentioned number of different experiments. ***P , 0.001;** P , 0.01, significantly different compared with control conditions,in the absence of hypoglycaemia, hypoxia, or ischaemia inductors;###P , 0.001; ##P , 0.01, significantly different compared withhypoglycaemic-like conditions.

364 Pereira et al.

Fig. 3. Effect of glutamate on the ability of PC12 cell to reduceMTT under metabolic inhibition conditions (hypoglycaemia,hypoxia, or ischaemia). Cells were plated and grown for 24 hrin culture medium (A) and, then hypoglycaemic (B), hypoxic(C), or ischaemic-like (D) conditions were induced as describedin Materials and Methods. Cells were then washed and switchedto fresh culture medium in the absence or in the presence of 0.5or 10 mM glutamate. After a 3-hr incubation, control and

glutamate-treated cells were analyzed for their ability to reducethe tetrazolium salt MTT. Data, expressed as the percentage (%)of control values determined under normoxic conditions, arethe arithmetic mean6 SEM of triplicate determinations of6–21 different experiments. ***P , 0.001; **P , 0.01; *P ,0.05, significantly different compared with control conditions,in the absence of glutamate.


tein in glucose-containing medium). A similar decrementof ATP levels was observed after incubation of PC12 cellsin glucose-free medium for 15, 20, or 30 min, incomparison with ATP levels determined after incubationin glucose medium during the same period of time.

Absence of NMDA Receptor Activation by Glutamatein PC12 Cells

Glutamate toxicity in PC12 cells was evaluated bydetermining the leakage of LDH after exposure to 100 or500 µM glutamate for 3 hr, in the presence or in theabsence of Mg21 (Table II). Despite a significant increasein the % of LDH released by the cells after incubation

with 500 µM glutamate, the % of release was similar inthe presence and in the absence of Mg21 (21.3296 3.81%and 24.16 2.93%, respectively). The simultaneous addi-tion of 5 µM MK-801 and 0.5 mM glutamate to Mg21-free medium did not decrease the release of LDH by thecells (Table II). Because short periods of exposure toglutamate concentrations in the micromole range aredescribed to activate the NMDA receptor and the toxiceffects mediated by receptor activation, cellular survivalwas evaluated after a 30-min incubation with 100 µMglutamate under the following conditions: presence ofglucose and Mg21, absence of glucose, absence of Mg21,or absence of both glucose and Mg21 (Table III). Underthese conditions, the % of LDH released was similar tothat determined in controls. To test the possible mecha-nism of delayed cell death induced by glutamate, the % ofLDH released was determined 24 hr after incubation ofPC12 cells with 100 µM glutamate under the experimen-tal conditions referred to above. Again, the % of deadcells was not significantly increased compared with thatdetermined in cells incubated in the absence of glutamate(Table III).

DISCUSSIONIn the present study we have shown that chemically

induced metabolic inhibition has different effects on theATP levels of PC12 cells. When cells are chemicallysubmitted to mitochondrial respiratory chain inhibition inthe presence of glucose (hypoxia), the capacity of mito-chondria dehydrogenases to reduce the tetrazolium saltMTT and the intracellular ATP levels are preserved (Fig.2 and Table I). Exposure of PC12 cells to glucosedeprivation (hypoglycaemia) resulted in a significantdecrease in ATP levels (Table I) without any alteration in

Fig. 4. Effect of antioxidants on the glutamate-induced inhibi-tion of the ability of PC12 cells to reduce MTT under metabolicstress conditions: hypoglycaemic (A) and ischaemic (B) condi-tions. Cells were plated and grown for 24 hr in culture mediumsupplemented with 10 µM vitamin E or 1 µM idebenone. Then,hypoglycaemic or ischaemic-like conditions were induced asdescribed in Materials and Methods. Cells were washed andswitched to fresh culture medium in the absence or in thepresence of 0.5 or 10 mM glutamate. After a 3-hr incubation,control and glutamate-treated cells were analyzed for theirability to reduce the tetrazolium salt MTT. Data obtained fromMTT assay, expressed as the percentage (%) of control valuesdetermined under normoxic conditions, are the arithmeticmean6 SEM of triplicate determinations of the mentioned numberof different experiments. ***P , 0.001; **P , 0.01; *P ,0.05, significantly different compared with control conditions, in theabsence of glutamate.111P , 0.001;11P , 0.01;1P ,0.05, significant different compared with glutamate-treatedcells, in the absence of antioxidants.

366 Pereira et al.

the MTT reduction by the cells (Fig. 2). However, underhypoxic plus hypoglycaemic conditions (ischaemia), asignificant inhibition of MTT reduction was observed(Fig. 2), and the ATP levels fell dramatically (Table I).Despite the decline in ATP levels, observed under hypo-glycaemic or ischaemic-like conditions, the plasma mem-brane integrity was preserved (data not shown). Further-more, we found that the major decrement of the energystatus of PC12 cells strongly potentiates the susceptibilityof the cells to glutamate (Fig. 3). Also, the increasedglutamate susceptibility upon metabolic inhibition seemsto be correlated with the intracellular ATP levels (Fig.

5A,B). The toxic effects of glutamate on metabolic stress,induced by hypoglycaemic or ischaemic-like conditions,seem to occur independently of NMDA receptor activa-tion (Tables II and III).

Viability of PC12 cells, determined by LDH leak-age as an index of plasma membrane integrity loss, inresponse to 30 min of chemical hypoxia, hypoglycaemia,or ischemia (hypoxia plus hypoglycaemia), remainedhigh (data not shown). This may indicate a delayedpattern of cell loss observed in many models of isch-aemia, where several hours of energy depletion arerequired before plasma membrane integrity is altered,despite the rapid and nearly complete ATP depletion(Eleff et al., 1991). Under hypoglycaemic and ischaemic-like conditions, a major reduction in the ATP levels of the

TABLE II. Effect of Mg 21 on the LDH Leakage Induced byGlutamate on PC12 Cells

[Glutamate](µM)

LDH Release (% of total)

1Mg21 2Mg21

0 10.1896 1.47 (n5 8) 13.636 2.64 (n5 6)100 12.7706 0.20 (n5 5) 11.936 1.17 (n5 6)500 21.3296 3.81 (n5 6)** 24.1 6 2.93 (n5 6)**500 plus 5 µM

MK-801ND 22.26 2.216 (n5 17)**

PC12 cells were incubated for 3 hr in Krebs buffer containing 100 or500 µM glutamate, in the presence of 10 µM glycine. Krebs buffercontained either zero or 1.0 mM Mg21. MK-801 (5 µM) was added tothe Mg21-free medium when cells were incubated for 3 hr with 500 µMglutamate. At the end of the incubation, the medium and the cells wereanalyzed for LDH activity. Data, expressed as the % of total activity,are the arithmetic mean6 SEM of triplicate determinations of thementioned number of different experiments.** P , 0.01, significantly different compared with control conditions,in the absence of glutamate.

Fig. 5. Influence of metabolic inhibition induced by hypogly-caemia on (A) the ability of glutamate-treated PC12 cell toreduce MTT and on (B) intracellular ATP levels. Cells wereplated and grown for 24 hr in culture medium, and thenhypoglycaemia was induced by incubating the cells for 5, 10,15, 20, or 30 min in a glucose free-medium. Extraction ofadenine nucleotides was performed immediately after incuba-tion of cells under hypoglycaemic like-conditions or in thepresence of glucose (control conditions), whereas for the MTTassay the cells were washed and switched to fresh culturemedium in the absence or in the presence of 0.5 or 10 mMglutamate. After a 3-hr incubation, control and glutamate-treated cells were analyzed for their ability to reduce thetetrazolium salt MTT. Data obtained from MTT assay, ex-pressed as the percentage (%) of control values, are thearithmetic mean6 SEM of triplicate determinations of thementioned number of different experiments. ***P , 0.001;** P , 0.01, significantly different compared with controlconditions.


cells was observed (Table I) compared with hypoxicconditions, indicating that glycolysis plays a very impor-tant role in regulating the energy status of PC12 cells.PC12 cells are neoplastic in nature and have a high rate ofglycolysis accompanied by a large production of lactateand a low use of glucose carbon through the Krebs cycle(Morelli et al., 1986). Thus, PC12 cells appear to havemultiple pathways capable of maintaining ATP produc-tion. Previous studies showed that PC12 cells maintainATP concentrations in the absence of oxygen if glucose ispresent (Gibson and Toral-Barza, 1990; Carroll et al.,1991). Although PC12 cells submitted to hypoxia did notshow significant alterations in the ATP levels, the reduc-tion in the energy status of the cells under ischaemic-likeconditions was shown to be potentiated in comparisonwith cells submitted to hypoglycaemia or hypoxia alone.This suggests that under hypoxia, although the synthesisof ATP in the mitochondria may be already compromised,glycolysis is stimulated, resulting in a maintenance of theATP levels, as observed in Table I. Therefore, eithermitochondrial respiratory alterations were subtle, allow-ing normal ATP production via oxidative phosphoryla-tion, or compensatory glycolytic ATP production wassufficient to fuel ATP recovery during mitochondrialdysfunction. The stimulation of glycolysis was alreadyproposed to occur in synaptosomes upon inhibition of therespiratory chain with cianide (Kauppinen and Nicholls,1986) and in retina cells upon chemical ischaemia (Regoet al., 1996). Recent studies also have reported thatglycolysis intermediates prevent hepatocyte injury in-duced by ‘‘chemical hypoxia,’’ by enhancing the produc-tion of glycolytic ATP (Gasbarrini et al., 1992). Theincrease in the rate of anaerobic glycolysis, to preservetissue ATP, also can occur in ischaemia preconditioning,by the mediation of glucose uptake or by the stimulationof key enzymes of the glycolytic pathway (Janier et al.,1994). On the other hand, lactate, the end product ofanaerobic glucose metabolism, could act as an alternativesubstrate for cells (Tsacopoulos and Magistretti, 1996).Following ischaemia, when the availability of glucose is

compromised, large releases of lactate are detected,reducing the neuronal loss (Pellerin and Magistretti,1994). During hypoglycaemia we observed a mainte-nance of the capacity of mitochondria dehydrogenases toreduce the tetrazolium salt MTT, although a significantdecrease in ATP occurs. Upon glucose deprivation (hypo-glycaemia) the inhibition of glycolysis may stimulatemitochondria to maintain ATP levels above a ‘‘criticalthreshold,’’ such that the activity of mitochondria dehydro-genases is not compromised. This also may be due, asproposed by Carroll et al. (1992), to the presence of otherenergy sources, such as glutamine, that can be used togenerate ATP, or even to the use of lactate as a readyenergy source after glucose depletion, as shown byBasma et al. (1992). The use of other energy sources byglycolysis to produce and to maintain ATP levels above a‘‘critical threshold’’ in our experimental conditions ofglucose deprivation is supported by the fact that whenPC12 cells were submitted to hypoglycaemia with iodo-acetic acid, an inhibitor of the glycolytic pathway, in thepresence of glucose, a significant decrease on MTTreduction was observed (data not shown).

The increase in glutamate potency after the declinein ATP levels induced by hypoglycaemic or ischaemic-like conditions is shown in Figure 3. When PC12 cellswere submitted to hypoglycaemia or ischaemia by chemi-cally inducing metabolic blockade of glycolysis or bothglycolysis and electron transport by the mitochondria,cellular exposure to glutamate for 3 hr resulted in asignificant decrease in the ability of cells to reduce MTT,more pronounced upon ischaemic-like conditions wheremore than 90% ATP was depleted. The increased gluta-mate susceptibility is well correlated with the decrease onATP levels (Fig. 5), suggesting that when PC12 cells aredepleted of intracellular ATP levels above a criticalthreshold, they are enabled to respond to toxic insults.Incubation of PC12 cells with both glutamate concentra-tions, during 3 hr, did not result in an alteration of MTTreduction by mitochondrial dehydrogenases (Fig. 1B)when cells were not previously exposed to metabolic

TABLE III. Effect of Mg 21 and Glucose on the LDH Leakage Induced by Glutamate

LDH release (% of total)

Control 100 µM glutamate

A B A B

5.6 mM glucose 1.0 mM Mg21 13.756 1.42 (n5 8) 17.756 1.66 (n5 8) 15.836 1.23 (n5 8) 17.336 0.63 (n5 8)5.6 mM glucose zero Mg21 14.36 2.11 (n5 4) 20.96 0.93 (n5 4) 14.686 1.89 (n5 9) 19.876 2.06 (n5 9)Zero glucose 1.0 mM Mg21 13.436 1.03 (n5 5) 19.986 1.57 (n5 7) 12.46 1.39 (n5 5) 20.06 2.23 (n5 5)Zero glucose zero Mg21 13.056 2.47 (n5 5) 20.876 2.19 (n5 5) 11.836 1.41 (n5 5) 20.136 0.51 (n5 5)

PC12 cells were incubated for 30 min in Krebs buffer containing 100 µM glutamate and 10 µM glycine. Krebs buffer contained either zero or 5.6mM glucose and/or zero or 1.0 mM Mg21. At the end of the incubation (A) or 24 hr after incubation in culture medium (B), the medium and thecells were analyzed for LDH activity. Data, expressed as the % of total activity, are the arithmetic mean6 SEM of triplicate determinations of thementioned number of different experiments.

368 Pereira et al.

stress. There are several reports in the literature showingthat metabolic inhibition (Cheng and Mattson, 1992;Greene et al., 1993; Patel et al., 1994; Maragos andSilverstein, 1995; Budd and Nicholls, 1996) enhances theneurotoxicity of glutamate. However, evidence suggeststhat the NMDA subtype of glutamate receptor, in particu-lar, is involved in mediating much of the neuronaldamage following focal ischaemia, hypoxia/anoxia, orhypoglycaemia (see Choi and Rothman, 1990, for re-view). Further evidence linking glutamate receptors toneuronal damage following inhibition of metabolism,comes from the observation that extracellular brain levelsof glutamate rise dramatically during ischaemia (Ben-veniste et al., 1984) and hypoglycaemia (Butcher et al.,1987), suggesting that it is this increase in extracellularglutamate that initiates glutamate receptor involvement inthe pathological process.

Zeevalk and Nicklas (1990) showed that the NMDAreceptor is activated and contributes, almost exclusively,to the initial acute excitotoxicity during mild metabolicstress induced by chemical inhibition of either glycolysisor electron transport, without any measurable rise inextracellular EAAs. During more severe metabolic stress,glutamate levels increase significantly, but this increaseoccurs only after NMDA receptor activation (Zeevalk andNicklas, 1991). One hypothesis for the mechanism bywhich receptor sensitivity to nontoxic glutamate concen-trations, during metabolic stress, may occur is by mitiga-tion of the voltage-dependent Mg21 blockade of theNMDA receptor channel caused by a loss of K1 homeo-stasis and/or deteriorating membrane potential. Henne-berry (1989) first proposed this mechanism, later con-firmed by Zeevalk and Nicklas (1992), to explain thefindings that incubation of cerebellar granule cell culturesin medium lacking glucose and Mg21 increased the lethaldose of glutamate. Depolarization of the plasma mem-brane evokes Ca21 influx through voltage-gated Ca21

channels and also through NMDA-gated Ca21 channels(Mayer and Westbrook, 1984; Nowak et al., 1984),leading to an increase in [Ca21]i. Intracellular calciumnormally serves important functions as a second messen-ger, metabolic regulator, and membrane stabilizer, andthen an excessive increase in [Ca21]i provokes cata-strophic enzymatic processes, leading to irreversibleneuronal injury (Siesjo¨ and Bengtsson, 1989).

According to previous reports, the results presentedhere (Tables II and III) clearly show that, under ourexperimental conditions, the NMDA receptor for gluta-mate is not involved in glutamate-induced toxicity, sug-gesting that glutamate toxicity in PC12 cells is notmediated by NMDA receptor activation (Schubert et al.,1992; Froissard and Duval, 1994). Recently, we showedthat glutamate is toxic to a PC12 cell line by inhibitingcystine uptake (Pereira and Oliveira, 1997). The depriva-

tion of cystine, an essential precursor for glutathionesynthesis, reduces GSH levels, and the cell is damaged byits inability to remove free radicals. The present studyshows that the loss of the voltage-dependent Mg21 blockat the NMDA receptor is not the only mechanismresponsible for the increased glutamate susceptibilityunder metabolic inhibition. However, the mechanism bywhich ATP depletion in PC12 cells creates the environ-ment in which glutamate becomes toxic is unknown.Elevated free cytosolic Ca21 does not appear to contrib-ute to the mechanisms of increased glutamate potency,because it was shown that, in the absence of glutamatereceptors (Johnson et al., 1994), the [Ca21]i increasesminimally during metabolic inhibition. One possibleexplanation for the susceptibility of PC12 cells to gluta-mate after ATP depletion is the production of reactiveoxygen species (ROS). In fact, the presence of ROS ininjured cells after treatment with metabolic inhibitors waspreviously shown (Gores et al., 1989; Yankuan et al.,1996). Under our experimental conditions, the antioxi-dants vitamin E and idebenone, when preincubated withthe cells and also present during the induction of thehypoglycaemic or ischaemic insults, greatly preventedthe inhibition of the redox activity induced by furtherexposure to glutamate (Fig. 4). These results suggest thatthe increased generation of free radicals during hypogly-caemia or ischaemia conditions is responsible for theenhanced susceptibility of PC12 cells to glutamate.

In conclusion, our data suggest that upon hypogly-caemia or ischaemia a decline in ATP occurs and PC12cells become susceptible to glutamate. This increasedsusceptibility seems to be dependent on the extent of ATPdepletion. After metabolic inhibition, glutamate toxicitypotentiation occurred via a mechanism that does notinvolve the activation of the NMDA glutamate receptorsubtype. In this process, the generation of ROS seems toplay a crucial role. The present data further highlight theimportance of preserving glycolysis and energy levels tomaintain normal cell function and protect cells fromdamaging insults, such as glutamate. The protectiveaction of antioxidants, against the increased susceptibilityto glutamate upon metabolic inhibition induced by hypo-glycaemic or ischaemic conditions in PC12 cells, sug-gests that antioxidants may slow the clinical progressionof the diseases in which oxidative stress and excessiveglutamate release (or deficient uptake) seem to be in-volved.

ACKNOWLEDGMENTS

The present work was supported by JNICT (Portu-guese Research Council) and the Human Capital MobilityProgram (EU), Proposal ERB 4050 PL 932039.


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Metabolic inhibition increases glutamate susceptibility on a PC12 cell line