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Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion
Brain Research 790 Ž1998. 209–216
Research report
Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion Angeles Almeida a , Simon J.R. Heales b , Juan P. Bolanos ˜ a
a, )
, Jose´ M. Medina
a
Departamento de Bioquımica y Biologıa ´ ´ Molecular, UniÕersidad de Salamanca, Salamanca, Spain b Department of Clinical Biochemistry, Institute of Neurology, London, UK Accepted 13 January 1998
Abstract The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief Ž15 min. exposure of the neurones to glutamate caused a dose-dependent Ž0.01–1 mM. increase in cyclic GMP levels together with delayed Ž24 h. neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide Ž PNO. synthase ŽNOS. inhibitor Nv-nitro-L-arginine methyl ester ŽNAME; 1 mM. or by the N-methyl-D-aspartate ŽNMDA. glutamate-subtype receptor antagonist D-Žy.-2-amino-5-phosphonopentanoate ŽAPV; 0.1 mM.. Glutamate exposure Ž0.1 mM and 1 mM. followed by 24 h of incubation caused the inhibition of succinate–cytochrome c reductase Ž20–25%. and cytochrome c oxidase Ž31%. activities in the surviving neurones, without affecting NADH–coenzyme-Q1 reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose Žby 26%. or succinate Žby 39%. as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion Ž20%. by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving PNO biosynthesis. q 1998 Elsevier Science B.V. Keywords: Glutamate; Neurotoxicity; Nitric oxide; Cell respiration; Mitochondria; Glutathione
1. Introduction Nitric oxide Ž PNO. is a neural messenger which is synthesized through the activation of Ca2q-dependent, neuronal PNO synthase ŽnNOS. after glutamate receptor stimulation w18x. Its ability to increase cyclic GMP ŽcGMP. concentration strongly suggests that PNO is involved in many important neurological functions Žfor a review, see, Ref. w31x.. However, under certain circumstances PNO biosynthesis may be enhanced, causing neurotoxicity w14x. The exacerbation of PNO biosynthesis within the brain may occur through two main different mechanisms. First, Abbreviations: APV, D-Žy.-2-amino-5-phosphopentanoate; cGMP, cyclic GMP; CoQ1, coenzyme Q1; DMEM, Dulbecco’s modified Eagle’s medium; EBSS, Earle’s balanced salts solution; Glu, L-glutamate; LDH, lactate dehydrogenase; NAME, Nv-nitro-L-arginine methyl ester; NOS, nitric oxide synthase ) Corresponding author. Departamento de Bioquımica y Biologıa ´ ´ Molecular, Universidad de Salamanca, Edificio Departamental, Avda. del Campo Charro, 37007 Salamanca, Spain. Fax: q34-23-29-45-79; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 6 4 - X
mediated by glutamate receptor over-stimulation, leading to the activation of nNOS in neurones w42x and, secondly, through cytokines-mediated induction of inducible NOS in glial cells w4,7,29,44,46x. At present, there is a large body of evidence to suggest that both pathways of enhancing P NO synthesis in the brain are involved in the neuronal death observed in several neurodegenerative diseases w5,41x. Glutamate-receptor stimulation in neurones causes a . w24x and a burst of superoxide anion formation ŽO Py 2 concomitant decrease in the activity of the Krebs cycle enzyme, aconitase w36x. Interestingly, the activity of purified aconitase is impaired by O Py and by the peroxynitrite 2 anion ŽONOOy. , but not by PNO w11,19x. These results suggests that NO species may not be responsible for neurotoxicity, but O 2 or, more convincingly, ONOOy formation from O Py and PNO w3x after glutamate-receptor 2 stimulation may account for its neurotoxicity w24x. Whilst the precise circumstances leading to neuronal death in neurodegenerative diseases is still a matter of debate, it is thought that energy depletion may be a key factor w2x. Furthermore, the mechanism of neuronal death by cy-
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tokine-dependent increase of glial iNOS activity has been proposed to be related by an PNO-mediated inhibition of the mitochondrial respiratory chain w7,8x. Exogenous ONOOy causes mitochondrial respiratory chain inhibition leading to neuronal cell death w6x, suggesting that mitochondrial dysfunction plays has a role on the mechanism leading to neurotoxicity due to excessive PNO biosynthesis. In addition, cellular antioxidant status, such as reduced glutathione ŽGSH. concentration protects neurones from ONOOy mediated mitochondrial damage and neurodegeneration w7x. Although a role for mitochondrial dysfunction in the mechanism of neurotoxicity through the glutamate– PNO pathway has been proposed w5,8x, no experimental evidence to support such hypothesis has so far been advanced. According to the mitochondrial susceptibility to excess P NOrONOOy formation w8,10,12,38x and the role of GSH at modulating NO-mediated cellular damage w6,7,37x, the aim of this study was to ascertain whether disruption, via NO, of neuronal mitochondrial function and GSH status are factors in glutamate neurotoxicity.
2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle’s medium ŽDMEM., Earle’s balanced salts solution ŽEBSS., fetal calf serum, horse serum and cytosine arabinoside were obtained from Sigma ŽSt. Louis, MO, USA.. Plastic tissue culture dishes were purchased from Nunc ŽDenmark.. L-Glutamate ŽGlu., glycine, Nv-nitro-L-arginine monomethyl ester ŽNAME. and ubiquinone-5 Žcoenzyme Q 1; CoQ1; catalog number C-7956. were obtained from Sigma and D-Žy.-2-amino5-phosphonopentanoate ŽAPV. from Research Biomedicals ŽNatick, MA, USA.. Other substrates, enzymes and coenzymes were purchased from Sigma or Boehringer ŽGermany.. Cytochrome c ŽBoehringer., was reduced with sodium ascorbate just before use and passed through Sephadex G-25M ŽPD-10 columns, Pharmacia, Uppsala, Sweden. to remove the excess ascorbate. 2.2. Animals Albino Wistar rats fed ad libitum on stock laboratory diet were used for the experiments. Rats were maintained at 238C with a 12 h light–dark cycle. Virgin females weighing 210–250 g were caged overnight with males and conception was confirmed the next morning by the presence of spermatozoa in vaginal smears. 2.3. Cell culture Cerebral cortex neurones in primary culture were prepared following the procedure described by Vicario et al.
w49x from fetal rats at 16–17 days of gestation, obtained by rapid hysterectomy after cervical dislocation of the mother. Dissociated cell suspensions were plated at a density of 2.1 = 10 5 cellsrcm2 in either 2 cm2 , 9.6 cm2 , 20 cm2 or 78.5 cm2 plastic Petri dishes coated with poly-D-lysine in DMEM supplemented with 10% fetal calf serum. Cells were incubated at 378C in a humidified atmosphere containing 5% CO 2r95% air. After 48 h, the medium was replaced with DMEM supplemented with 5% horse serum and 20 mM glucose. At day 4 of culture, cytosine arabinoside was added to a final concentration of 10 m M in order to prevent astrocyte proliferation w49x and neurones were used at day 9. 2.4. Exposure of neurones to glutamate For the exposure of neurones to glutamate, culture medium was removed and neurones were washed once with prewarmed Žf 378C. buffered Hanks’ solution Ž5.26 mM KCl, 0.43 mM KH 2 PO4 , 132.4 mM NaCl, 4.09 mM NaHCO 3 , 0.33 mM Na 2 HPO4 , 5.44 mM glucose, 2 mM CaCl 2 , and 20 mM HEPES, pH 7.4.. After 5 min of preincubation in the absence or presence of NAME Ž1 mM. or APV Ž0.1 mM., L-glutamate was added from concentrated solutions to the final concentration indicated Ž0.01–1 mM. Žplus 10 m M glycine.. Neurones were incubated at 378C for 15 min, the buffer was aspirated, replaced with DMEM plus 5% horse serum and the cells were incubated at 378C for further 24 h in the absence of effectors. 2.5. Enzyme determinations For the determination of the mitochondrial respiratory chain complex activities, neurones plated in 78.5 cm2 Petri dishes were used. Following the glutamate exposure and the 24 h incubation period as described above, neuronal cultures were washed with cold EBSS Žwithout Ca2q and Mg 2q . and the surviving cells were collected by trypsinization and resuspended in 0.5 ml of a buffer containing 320 mM sucrose, 1 mM EDTA and 10 mM Tris–HCl at pH 7.4. Cell suspensions Žcontaining about 3–4 mg of proteinrml. were frozen and thawed three times to ensure cell lysis. Enzyme activities were determined in the cell lysates using a Hitachi U2000 spectrophotometer ŽHitachi, Tokio, Japan.. NADH-1 reductase Žcomplex I; EC 1.6.99.3. activity was measured as described in Ragan et al. w39x. The activity of succinate–cytochrome c reductase Žcomplex II–III; EC 1.8.3.1. was determined following the method of King w22x. Cytochrome c oxidase Žcomplex IV; EC 1.9.3.1. activity was determined as described by Wharton and Tzagoloff w51x. Citrate synthase activity ŽEC 4.1.3.7. was measured after Shepherd and Garland w43x. For the determination of lactate dehydrogenase ŽLDH, EC 1.1.1.27. activity, the culture medium was collected after the 24 h incubation period
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ked oxygen consumption was completely abolished with antimycin Ž5 m grml.. The registered rates of oxygen consumption were used to calculate the slopes, and they were expressed as nanogram-atoms of oxygen consumed per minute per milligram of protein. 2.7. Metabolite determinations
Fig. 1. Dose response curves for glutamate mediated Ža. cGMP formation, Žb. LDH release and Žc. ATP concentration in neurones in primary culture. Neurones were exposed for 15 min to increasing glutamate concentrations and the cells were incubated for further 24 h in the absence of glutamate. Cellular cGMP and ATP concentrations and LDH activity released to the culture medium were measured as described in Section 2. The correlation between LDH activity and ATP concentration is shown in panel d. Results are the mean"S.E.M. values from four to five different culture preparations.
For cGMP determinations, neurones plated in 9.6 cm2 wells were exposed to glutamate essentially as described above, except that 1-isobutyl-3-methylxanthine Ž1 mM. was included throughout the experiment. After the 15 min incubation period, the buffer was aspirated and cells were scraped with 2 = 0.5 ml of ice-cold ethanol. After evaporation of the solvent, cell extracts were used for cGMP determination using a commercially available radioimmunoassay kit ŽAmersham, UK. following the manufacturer’s instructions. For ATP determinations, neurones plated in 2 cm2 wells were exposed to glutamate as described above. After the 24 h incubation period, cells were quickly washed with ice-cold EBSS Žwithout Ca2q and Mg 2q ., scraped with 2 = 0.5 ml of 0.3 M HClO4 neutralized with 0.5 ml of 2 M KHCO 3 to pH 6.5 and centrifuged. ATP was determined in the supernatants by
and placed in ice until use within the same day. LDH activity was measured following the method of Vassault w48x. LDH release to medium was used as an index of the cell death as previously described w23x. All enzyme activities were expressed as nanomoles per minute per milligram of protein, except for cytochrome c oxidase, which was expressed as the first-order rate constant Ž k miny1 mgy1 of protein.. 2.6. Oxygen consumption experiments For the measurement of oxygen consumption rates, neurones plated in 78.5 cm2 Petri dishes were used. Following the glutamate exposure and the 24 h incubation period as described above, cells were collected by trypsinization, rinsed once with buffered Hanks’ solution and resuspended in 0.5 ml of buffered Hanks’ solution Žwithout glucose., giving a final concentration of about 9 mg of proteinrml. Cell suspensions were kept in ice until used for oxygen consumption measurements Žwithin 1 h.. This was performed using a 2 ml capacity magnetically stirred incubation chamber with a water jacket and a Clark-type electrode ŽGilson, Medical Electronic, France.. A 0.2 ml aliquots of the cell suspension were incubated in the chamber at 308C with 1.8 ml of buffered Hanks’ solution and the rate of oxygen consumption was monitored. Overall cell oxygen consumption was estimated using glucose Ž5 mM. as the substrate. After inhibition of NADH-linked oxygen consumption with rotenone Ž10 m M., succinate Ž10 mM. was added in order to monitor the rates of FADH-linked oxygen consumption. FADH-lin-
Fig. 2. The effect of NAME and APV on glutamate-mediated Ža. cGMP formation, Žb. LDH release and Žc. ATP concentration in neurones in primary culture. Neurones were exposed, or not, to glutamate Ž0.1 or 1 mM. for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. Cellular cGMP and ATP concentrations and LDH activity released to the culture medium were measured as described in Section 2. Results are the mean"S.E.M. values from four to five different culture preparations. )Significantly different when compared to control group.
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Table 1 Effect of glutamate exposure on the activity of the mitochondrial respiratory chain complexes in neurones in primary culture Condition
NADH–CoQ1 reductase Žnmolrminrmg.
Succinate cytochrome c reductase Žnmolrminrmg.
Cytochrome c oxidase Žkrminrmg.
Control Glu 0.1 mM Glu 0.1 mM q NAME Glu 0.1 mM q APV Glu 1 mM Glu 1 mM q NAME Glu 1 mM q APV
35.5 " 1.69 34.2 " 2.48 36.8 " 1.01 34.9 " 2.05 31.9 " 2.62 35.5 " 3.23 35.2 " 2.54
17.3 " 0.56 13.7 " 1.47 a 17.8 " 0.58 16.2 " 1.01 13.0 " 0.67 a 16.6 " 0.91 15.2 " 1.56
2.01 " 0.16 1.91 " 0.10 2.19 " 0.05 1.90 " 0.09 1.39 " 0.06 a 2.16 " 0.15 1.83 " 0.04
Neurones were exposed to glutamate Ž0.1 or 1 mM. for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for a further 24 h in the absence of effectors. Enzyme activities were determined in the surviving cells as described in Section 2. Values are mean " S.E.M. from four to five different culture preparations. a Significantly different when compared to control group.
chemiluminescence using a commercially available kit ŽSigma. following the manufacturer’s instructions. For GSH determination, aliquots of the cell lysates were treated with 15 mM o-phosphoric acid and centrifuged. GSH concentration was determined in the supernatants by HPLC using electrochemical detection w40x. 2.8. Protein determination Proteins were determined either in the cell suspensions, lysates or in parallel cell culture incubations after solubilization with 0.1 M NaOH. Protein concentration was determined by the method of Lowry et al. w27x. 2.9. Statistical analysis Measurements from individual cultures were performed in duplicate and results are expressed as the mean " S.E.M. values for the number of culture preparations indicated. Statistical analysis of the results was determined by oneway analysis of variance followed by the least significant difference multiple range test. In all cases, p - 0.05 was considered significant.
negative correlation Ž r s 0.87. with LDH released ŽFig. 1d.. Glutamate stimulation of cGMP levels was antagonized with NAME Ž1 mM., a potent inhibitor of NOS, and with APV Ž0.1 mM., a competitive antagonist of NMDA glutamate-subtype receptor w17x ŽFig. 2a.. Similarly, either NAME or APV prevented both the increase in LDH release ŽFig. 2b. and the ATP depletion ŽFig. 2c. caused by glutamate. We have previously described that neuronal mitochondrial respiratory chain is damaged following exposure to either ONOOy or PNO donors w6,7x. Accordingly, we were prompted to investigate whether the glutamate-mediated loss of ATP content found was related to the impairment of mitochondrial function. As shown in Table 1, the exposure to glutamate Ž0.1 mM or 1 mM. decreased, by about 20–25%, the activity of succinate cytochrome c reductase, although NADH–CoQ reductase activity remained unchanged in the surviving neurones. The activity of cytochrome c oxidase was significantly decreased Ž31%. when neurones were exposed to 1 mM glutamate, but unchanged by 0.1 mM glutamate. Citrate synthase activity was unaffected by the treatment Žin nmolrminrmg of protein, control, 137.1 " 7.04, n s 4; 1 mM glutamate,
3. Results It is accepted that the increase of cGMP may be regarded as an index of endogenous NO formation w18x. Thus, our results show that a brief Ž15 min. exposure of neurones to glutamate resulted in a dose-dependent increase in cGMP concentration to about 0.05 mM glutamate ŽFig. 1a.. Increasing glutamate concentration further did not result in higher cGMP concentration ŽFig. 1a., possibly reflecting maximal guanylate cyclase activity. In addition, glutamate exposure caused neurotoxicity as shown by the increase of LDH released to the culture medium w23x; the phenomenon was dose-dependent and delayed Ž24 h. ŽFig. 1b.. This effect was accompanied by a decrease in neuronal ATP concentration ŽFig. 1c., showing a significant
Table 2 Effect of glutamate exposure on neuronal respiration Condition
Oxygen consumption Žng atoms Orminrmg. Glucose
Succinate
Control Glu 1 mM Glu 1 mMqNAME Glu 1 mMqAPV
6.61"0.39 4.89"0.32 a 6.11"0.56 5.99"0.53
7.10"0.67 4.35"0.36 a 7.65"0.61 6.68"0.89
Neurones were exposed to 1 mM glutamate for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. Neuronal respiration was measured as described in Section 2 Žsee Fig. 3.. Results are the mean"S.E.M. values from three different culture preparations. a Significantly different when compared to control group.
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gen consumption; 39% of inhibition.. These effects were prevented by NAME or APV ŽTable 2.. A diagrammed run for each condition is shown in Fig. 3. GSH is an important intracellular defence against no mediated mitochondrial damage in neurones w6,7x. In view of this, we have determined GSH concentration in the neurones exposed to glutamate. Fig. 3 shows that a brief Ž15 min. glutamate exposure to the neurones significantly decreased GSH concentration by 20% after 24 h of incubation. This fall in GSH concentration caused by glutamate exposure was prevented by either inhibiting NOS with NAME or by antagonizing NMDA receptor glutamate-subtype with APV ŽFig. 4.. Fig. 3. Oxygen consumption rates in neuronal suspensions under several conditions. Neurones were exposed, or not, to 1 mM glutamate for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. Overall neuronal respiration was measured in cells resuspended in buffered Hanks’ solution with glucose Ž5 mM. as substrate. After inhibition of NADH-linked oxygen consumption with rotenone Ž10 m M., the rate of FADH-linked oxygen consumption Žantimycin-sensitive. was monitored with succinate Ž10 mM. as substrate. Mean slopes values for each condition are shown in Table 2.
135.1 " 8.0, n s 4.. The decrease in succinate cytochrome c reductase and cytochrome c oxidase activities caused by glutamate was completely prevented by NAME or by APV ŽTable 1.. In order to study whether glutamate-mediated inhibition of the respiratory chain components would affect to the respiratory function of the mitochondria, we measured the rate of oxygen consumption by the neurones exposed to glutamate. A brief Ž15 min. glutamate exposure to the neurones decreased, after the 24 h incubation period, the rates of respiration using both glucose Ž26% of inhibition. or succinate plus rotenone Žto inhibit NADH-linked oxy-
Fig. 4. The effect of glutamate exposure on GSH concentration and the protective effects of NAME and APV in neurones in primary culture. Neurones were exposed, or not, to glutamate Ž0.1 or 1 mM. for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. GSH concentration was measured in the cell lysates as described in Section 2. Results are the mean"S.E.M. values from four to five different culture preparations. )Significantly different when compared to control group.
4. Discussion Our results showing the dose-dependent, PNO-mediated increase in cGMP by glutamate exposure ŽFigs. 1 and 2. in neurones confirm those by others w18x and suggest that the neuronal cultures used in the present study may be considered as ‘mature’ and hence appropriate for studies involving glutamate neurotoxicity w15x. In addition, the brief glutamate exposure caused delayed PNO-mediated neurotoxicity, an effect that has been observed both in vitro w15x and in vivo w42x. The dose-dependent decrease in neuronal ATP concentration significantly correlated with cell death ŽFig. 1d., suggesting that impairment of energy metabolism may be a phenomenon related to glutamate-mediated neurotoxicity. This is in agreement with the study of Tsuji et al. w47x, who reported a rapid and irreversible decrease in spinal neuronal ATP by glutamate exposure. However, we have expanded this observation and show that ATP depletion is prevented by NAME ŽFig. 2c., strongly suggesting that neuronal energy impairment leading to neurotoxicity caused by glutamate-receptor stimulation is a process mediated by NOS activity. Several hypotheses involving a possible energy impairment have been postulated to explain the mechanism of glutamate-mediated PNO neurotoxicity. Thus, glutamate receptor stimulation causes activation of polyŽADP ribose. synthetase DNA-repair system, which requires ATP and hence might lead to neuronal energy deficiency w53x. However, blockade of polyŽADP ribose. synthetase activation has never been shown to prevent glutamate receptor-mediated ATP depletion in neurones and, therefore, the relevance of this mechanism for glutamate–NO neurotoxicity remains elusive. Another hypothesis involves the NO-mediated inhibition of the activity of the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase w30,54x. However, it has been shown that in these circumstances the extent of the inhibition is not sufficient to compromise cellular energy production w16,20x. Mitochondria have been considered to be a possible target for glutamate neurotoxicity, because calcium accumulation within this organelle has been observed after
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short exposures to glutamate w50,52x. However, the possible implication of PNO in these phenomena was not investigated. In the present work, we show that a brief glutamate exposure to the neurones caused a significant impairment of mitochondrial succinate–cytochrome c reductase Ž20.6% and 24.4% of inhibition at 0.1 mM and 1 mM glutamate, respectively. and cytochrome c oxidase Ž30.5% of inhibition at 1 mM glutamate. activities, without affecting NADH–CoQ1 reductase activity ŽTable 1.. These effects were completely prevented by APV or by NAME, suggesting that the inhibitory effects were mediated by the pathway involving NMDA-receptor stimulation leading to increased NOS activity. In addition, our results suggest that the inhibition of the respiratory chain by glutamate is irreversible, because it is observed after 24 h of incubation without glutamate and is not reversed by the collection, homogenization and dilution of the samples. Furthermore, this mitochondrial damage caused by glutamate impaired oxygen consumption ŽTable 2. and depleted ATP in the neurones ŽFig. 1c, Fig. 2.. Since the mitochondrial function parameters ŽFig. 1c, Fig. 2c; Tables 1 and 2. were determined only in the surviving neurones, our results strongly suggest that the observed mitochondrial damage preceded neuronal death. Glutamate-receptor stimulation causes a burst of Oy 2 formation w24x, suggesting that the endogenous synthesis of ONOOy under our conditions may be achieved. If so, the resulting ONOOy may be responsible for the direct, irreversible damage to succinate–cytochrome c reductase, which occurs even at very low glutamate concentration ŽTable 1.. This is in agreement with previous observation using authentic ONOOy in either isolated brain mitochondria w6x or submitochondrial particles w26x. Irreversible inhibition of cytochrome c oxidase activity after glutamate exposure ŽTable 1. has been observed in astrocytes w8x after iNOS induction, or in neurones w6x and isolated heart mitochondria w38x after ONOOy exposure. The exact mechanism causing irreversible cytochrome c oxidase inhibition by ONOOy is unknown. However, ONOOy is a well-known lipid pro-oxidant w37x and cardiolipin integrity is absolutely necessary for cytochrome c oxidase activity w45x. Since inhibition of cytochrome c oxidase activity is prevented by the antioxidant trolox w21x, it might be suggested that irreversible damage to cytochrome c oxidase by glutamate exposure could be carried out indirectly, possibly though ONOOy-mediated cardiolipin peroxidation. Finally, the lack of effect of glutamate exposure on neuronal NADH–CoQ reductase activity ŽTable 1. is consistent with the lack of effect of authentic ONOOy on this enzyme activity in neuronal cultures w6x, though other laboratories have observed a direct effect of ONOOy on respiration at NADH-linked substrates in submitochondrial particles w26x. This discrepancy can be explained by the preservation of the antioxidant status in the intact neurone. In fact, glutamate exposure to neurones has been shown to be accompanied by GSH depletion w34x and increased
oxidative stress. Furthermore, under our experimental conditions, GSH depletion by glutamate exposure was prevented by NAME ŽFig. 3., suggesting that NOS activity is involved in the effect. Whether GSH depletion is due to P NO-dependent inhibition of cystine uptake w33x or to a putative reaction of GSH with ONOOy w37x is unknown. In any case, the antioxidant reserve of neurones seems to be compromised under this situation, a fact that may favor the mitochondrial susceptibility to oxidative stress. The pathophysiological relevance of mitochondrial impairment caused by excessive synthesis of PNO in the CNS is well-accepted. Thus, neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, Hungtinton’s diseases or multiple sclerosis are accompanied by the impairment of the mitochondrial function w2,5,35x. Whilst a role for inducible NOS in glial cells have been ascribed in the pathology of these disorders w32x, there is also evidence suggesting that excessive glutamate-receptor stimulation may be involved in the neurodegenerative process w13,28x. Moreover, necrosis and apoptosis are associated with impairment of mitochondrial function. Thus, severe mitochondrial damage has been associated with necrosis, while a remanent mitochondrial function is required for an active apoptosis w1,9,25x. The present results showing that glutamate-receptor stimulation causes an dependent irreversible inhibition of mitochondrial respiration strongly supports the hypothesis that cellular energy deficiency might be a key factor in glutamate neurotoxicity and neurodegeneration. Acknowledgements A.A. is a recipient of an ‘Acciones de Reincorporacion ´ de Doctores y Tecnologos del M.E.C.’, Spain. J.P.B. and ´ S.J.R.H. are in receipt of a Biomedical Collaboration Grant from the Wellcome Trust, U.K. This work was supported by the F.I.S.S.S. and D.G.I.C.Y.T. ŽJ.M.M.. and by the Junta de Castilla y Leon ´ and C.I.C.Y.T. ŽJ.P.B.., Spain. References w1x M. Ankarcrona, J.M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S.A. Lipton, P. Nicotera, Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron 15 Ž1995. 961–973. w2x M.F. Beal, B.T. Hyman, W. Koroshetz, Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases?, Trends Neurosci. 16 Ž1993. 125–131. w3x J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall, B.A. Freeman, Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 1620–1624. w4x K.M. Boje, P.K. Arora, Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death, Brain Res. 587 Ž1992. 250–256. w5x J.P. Bolanos, ˜ A. Almeida, V. Stewart, S. Peuchen, J.M. Land, J.B. Clark, S.J.R. Heales, Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases, J. Neurochem. 68 Ž1997. 2227–2240.
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Research report
Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion Angeles Almeida a , Simon J.R. Heales b , Juan P. Bolanos ˜ a
a, )
, Jose´ M. Medina
a
Departamento de Bioquımica y Biologıa ´ ´ Molecular, UniÕersidad de Salamanca, Salamanca, Spain b Department of Clinical Biochemistry, Institute of Neurology, London, UK Accepted 13 January 1998
Abstract The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief Ž15 min. exposure of the neurones to glutamate caused a dose-dependent Ž0.01–1 mM. increase in cyclic GMP levels together with delayed Ž24 h. neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide Ž PNO. synthase ŽNOS. inhibitor Nv-nitro-L-arginine methyl ester ŽNAME; 1 mM. or by the N-methyl-D-aspartate ŽNMDA. glutamate-subtype receptor antagonist D-Žy.-2-amino-5-phosphonopentanoate ŽAPV; 0.1 mM.. Glutamate exposure Ž0.1 mM and 1 mM. followed by 24 h of incubation caused the inhibition of succinate–cytochrome c reductase Ž20–25%. and cytochrome c oxidase Ž31%. activities in the surviving neurones, without affecting NADH–coenzyme-Q1 reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose Žby 26%. or succinate Žby 39%. as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion Ž20%. by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving PNO biosynthesis. q 1998 Elsevier Science B.V. Keywords: Glutamate; Neurotoxicity; Nitric oxide; Cell respiration; Mitochondria; Glutathione
1. Introduction Nitric oxide Ž PNO. is a neural messenger which is synthesized through the activation of Ca2q-dependent, neuronal PNO synthase ŽnNOS. after glutamate receptor stimulation w18x. Its ability to increase cyclic GMP ŽcGMP. concentration strongly suggests that PNO is involved in many important neurological functions Žfor a review, see, Ref. w31x.. However, under certain circumstances PNO biosynthesis may be enhanced, causing neurotoxicity w14x. The exacerbation of PNO biosynthesis within the brain may occur through two main different mechanisms. First, Abbreviations: APV, D-Žy.-2-amino-5-phosphopentanoate; cGMP, cyclic GMP; CoQ1, coenzyme Q1; DMEM, Dulbecco’s modified Eagle’s medium; EBSS, Earle’s balanced salts solution; Glu, L-glutamate; LDH, lactate dehydrogenase; NAME, Nv-nitro-L-arginine methyl ester; NOS, nitric oxide synthase ) Corresponding author. Departamento de Bioquımica y Biologıa ´ ´ Molecular, Universidad de Salamanca, Edificio Departamental, Avda. del Campo Charro, 37007 Salamanca, Spain. Fax: q34-23-29-45-79; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 6 4 - X
mediated by glutamate receptor over-stimulation, leading to the activation of nNOS in neurones w42x and, secondly, through cytokines-mediated induction of inducible NOS in glial cells w4,7,29,44,46x. At present, there is a large body of evidence to suggest that both pathways of enhancing P NO synthesis in the brain are involved in the neuronal death observed in several neurodegenerative diseases w5,41x. Glutamate-receptor stimulation in neurones causes a . w24x and a burst of superoxide anion formation ŽO Py 2 concomitant decrease in the activity of the Krebs cycle enzyme, aconitase w36x. Interestingly, the activity of purified aconitase is impaired by O Py and by the peroxynitrite 2 anion ŽONOOy. , but not by PNO w11,19x. These results suggests that NO species may not be responsible for neurotoxicity, but O 2 or, more convincingly, ONOOy formation from O Py and PNO w3x after glutamate-receptor 2 stimulation may account for its neurotoxicity w24x. Whilst the precise circumstances leading to neuronal death in neurodegenerative diseases is still a matter of debate, it is thought that energy depletion may be a key factor w2x. Furthermore, the mechanism of neuronal death by cy-
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tokine-dependent increase of glial iNOS activity has been proposed to be related by an PNO-mediated inhibition of the mitochondrial respiratory chain w7,8x. Exogenous ONOOy causes mitochondrial respiratory chain inhibition leading to neuronal cell death w6x, suggesting that mitochondrial dysfunction plays has a role on the mechanism leading to neurotoxicity due to excessive PNO biosynthesis. In addition, cellular antioxidant status, such as reduced glutathione ŽGSH. concentration protects neurones from ONOOy mediated mitochondrial damage and neurodegeneration w7x. Although a role for mitochondrial dysfunction in the mechanism of neurotoxicity through the glutamate– PNO pathway has been proposed w5,8x, no experimental evidence to support such hypothesis has so far been advanced. According to the mitochondrial susceptibility to excess P NOrONOOy formation w8,10,12,38x and the role of GSH at modulating NO-mediated cellular damage w6,7,37x, the aim of this study was to ascertain whether disruption, via NO, of neuronal mitochondrial function and GSH status are factors in glutamate neurotoxicity.
2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle’s medium ŽDMEM., Earle’s balanced salts solution ŽEBSS., fetal calf serum, horse serum and cytosine arabinoside were obtained from Sigma ŽSt. Louis, MO, USA.. Plastic tissue culture dishes were purchased from Nunc ŽDenmark.. L-Glutamate ŽGlu., glycine, Nv-nitro-L-arginine monomethyl ester ŽNAME. and ubiquinone-5 Žcoenzyme Q 1; CoQ1; catalog number C-7956. were obtained from Sigma and D-Žy.-2-amino5-phosphonopentanoate ŽAPV. from Research Biomedicals ŽNatick, MA, USA.. Other substrates, enzymes and coenzymes were purchased from Sigma or Boehringer ŽGermany.. Cytochrome c ŽBoehringer., was reduced with sodium ascorbate just before use and passed through Sephadex G-25M ŽPD-10 columns, Pharmacia, Uppsala, Sweden. to remove the excess ascorbate. 2.2. Animals Albino Wistar rats fed ad libitum on stock laboratory diet were used for the experiments. Rats were maintained at 238C with a 12 h light–dark cycle. Virgin females weighing 210–250 g were caged overnight with males and conception was confirmed the next morning by the presence of spermatozoa in vaginal smears. 2.3. Cell culture Cerebral cortex neurones in primary culture were prepared following the procedure described by Vicario et al.
w49x from fetal rats at 16–17 days of gestation, obtained by rapid hysterectomy after cervical dislocation of the mother. Dissociated cell suspensions were plated at a density of 2.1 = 10 5 cellsrcm2 in either 2 cm2 , 9.6 cm2 , 20 cm2 or 78.5 cm2 plastic Petri dishes coated with poly-D-lysine in DMEM supplemented with 10% fetal calf serum. Cells were incubated at 378C in a humidified atmosphere containing 5% CO 2r95% air. After 48 h, the medium was replaced with DMEM supplemented with 5% horse serum and 20 mM glucose. At day 4 of culture, cytosine arabinoside was added to a final concentration of 10 m M in order to prevent astrocyte proliferation w49x and neurones were used at day 9. 2.4. Exposure of neurones to glutamate For the exposure of neurones to glutamate, culture medium was removed and neurones were washed once with prewarmed Žf 378C. buffered Hanks’ solution Ž5.26 mM KCl, 0.43 mM KH 2 PO4 , 132.4 mM NaCl, 4.09 mM NaHCO 3 , 0.33 mM Na 2 HPO4 , 5.44 mM glucose, 2 mM CaCl 2 , and 20 mM HEPES, pH 7.4.. After 5 min of preincubation in the absence or presence of NAME Ž1 mM. or APV Ž0.1 mM., L-glutamate was added from concentrated solutions to the final concentration indicated Ž0.01–1 mM. Žplus 10 m M glycine.. Neurones were incubated at 378C for 15 min, the buffer was aspirated, replaced with DMEM plus 5% horse serum and the cells were incubated at 378C for further 24 h in the absence of effectors. 2.5. Enzyme determinations For the determination of the mitochondrial respiratory chain complex activities, neurones plated in 78.5 cm2 Petri dishes were used. Following the glutamate exposure and the 24 h incubation period as described above, neuronal cultures were washed with cold EBSS Žwithout Ca2q and Mg 2q . and the surviving cells were collected by trypsinization and resuspended in 0.5 ml of a buffer containing 320 mM sucrose, 1 mM EDTA and 10 mM Tris–HCl at pH 7.4. Cell suspensions Žcontaining about 3–4 mg of proteinrml. were frozen and thawed three times to ensure cell lysis. Enzyme activities were determined in the cell lysates using a Hitachi U2000 spectrophotometer ŽHitachi, Tokio, Japan.. NADH-1 reductase Žcomplex I; EC 1.6.99.3. activity was measured as described in Ragan et al. w39x. The activity of succinate–cytochrome c reductase Žcomplex II–III; EC 1.8.3.1. was determined following the method of King w22x. Cytochrome c oxidase Žcomplex IV; EC 1.9.3.1. activity was determined as described by Wharton and Tzagoloff w51x. Citrate synthase activity ŽEC 4.1.3.7. was measured after Shepherd and Garland w43x. For the determination of lactate dehydrogenase ŽLDH, EC 1.1.1.27. activity, the culture medium was collected after the 24 h incubation period
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ked oxygen consumption was completely abolished with antimycin Ž5 m grml.. The registered rates of oxygen consumption were used to calculate the slopes, and they were expressed as nanogram-atoms of oxygen consumed per minute per milligram of protein. 2.7. Metabolite determinations
Fig. 1. Dose response curves for glutamate mediated Ža. cGMP formation, Žb. LDH release and Žc. ATP concentration in neurones in primary culture. Neurones were exposed for 15 min to increasing glutamate concentrations and the cells were incubated for further 24 h in the absence of glutamate. Cellular cGMP and ATP concentrations and LDH activity released to the culture medium were measured as described in Section 2. The correlation between LDH activity and ATP concentration is shown in panel d. Results are the mean"S.E.M. values from four to five different culture preparations.
For cGMP determinations, neurones plated in 9.6 cm2 wells were exposed to glutamate essentially as described above, except that 1-isobutyl-3-methylxanthine Ž1 mM. was included throughout the experiment. After the 15 min incubation period, the buffer was aspirated and cells were scraped with 2 = 0.5 ml of ice-cold ethanol. After evaporation of the solvent, cell extracts were used for cGMP determination using a commercially available radioimmunoassay kit ŽAmersham, UK. following the manufacturer’s instructions. For ATP determinations, neurones plated in 2 cm2 wells were exposed to glutamate as described above. After the 24 h incubation period, cells were quickly washed with ice-cold EBSS Žwithout Ca2q and Mg 2q ., scraped with 2 = 0.5 ml of 0.3 M HClO4 neutralized with 0.5 ml of 2 M KHCO 3 to pH 6.5 and centrifuged. ATP was determined in the supernatants by
and placed in ice until use within the same day. LDH activity was measured following the method of Vassault w48x. LDH release to medium was used as an index of the cell death as previously described w23x. All enzyme activities were expressed as nanomoles per minute per milligram of protein, except for cytochrome c oxidase, which was expressed as the first-order rate constant Ž k miny1 mgy1 of protein.. 2.6. Oxygen consumption experiments For the measurement of oxygen consumption rates, neurones plated in 78.5 cm2 Petri dishes were used. Following the glutamate exposure and the 24 h incubation period as described above, cells were collected by trypsinization, rinsed once with buffered Hanks’ solution and resuspended in 0.5 ml of buffered Hanks’ solution Žwithout glucose., giving a final concentration of about 9 mg of proteinrml. Cell suspensions were kept in ice until used for oxygen consumption measurements Žwithin 1 h.. This was performed using a 2 ml capacity magnetically stirred incubation chamber with a water jacket and a Clark-type electrode ŽGilson, Medical Electronic, France.. A 0.2 ml aliquots of the cell suspension were incubated in the chamber at 308C with 1.8 ml of buffered Hanks’ solution and the rate of oxygen consumption was monitored. Overall cell oxygen consumption was estimated using glucose Ž5 mM. as the substrate. After inhibition of NADH-linked oxygen consumption with rotenone Ž10 m M., succinate Ž10 mM. was added in order to monitor the rates of FADH-linked oxygen consumption. FADH-lin-
Fig. 2. The effect of NAME and APV on glutamate-mediated Ža. cGMP formation, Žb. LDH release and Žc. ATP concentration in neurones in primary culture. Neurones were exposed, or not, to glutamate Ž0.1 or 1 mM. for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. Cellular cGMP and ATP concentrations and LDH activity released to the culture medium were measured as described in Section 2. Results are the mean"S.E.M. values from four to five different culture preparations. )Significantly different when compared to control group.
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Table 1 Effect of glutamate exposure on the activity of the mitochondrial respiratory chain complexes in neurones in primary culture Condition
NADH–CoQ1 reductase Žnmolrminrmg.
Succinate cytochrome c reductase Žnmolrminrmg.
Cytochrome c oxidase Žkrminrmg.
Control Glu 0.1 mM Glu 0.1 mM q NAME Glu 0.1 mM q APV Glu 1 mM Glu 1 mM q NAME Glu 1 mM q APV
35.5 " 1.69 34.2 " 2.48 36.8 " 1.01 34.9 " 2.05 31.9 " 2.62 35.5 " 3.23 35.2 " 2.54
17.3 " 0.56 13.7 " 1.47 a 17.8 " 0.58 16.2 " 1.01 13.0 " 0.67 a 16.6 " 0.91 15.2 " 1.56
2.01 " 0.16 1.91 " 0.10 2.19 " 0.05 1.90 " 0.09 1.39 " 0.06 a 2.16 " 0.15 1.83 " 0.04
Neurones were exposed to glutamate Ž0.1 or 1 mM. for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for a further 24 h in the absence of effectors. Enzyme activities were determined in the surviving cells as described in Section 2. Values are mean " S.E.M. from four to five different culture preparations. a Significantly different when compared to control group.
chemiluminescence using a commercially available kit ŽSigma. following the manufacturer’s instructions. For GSH determination, aliquots of the cell lysates were treated with 15 mM o-phosphoric acid and centrifuged. GSH concentration was determined in the supernatants by HPLC using electrochemical detection w40x. 2.8. Protein determination Proteins were determined either in the cell suspensions, lysates or in parallel cell culture incubations after solubilization with 0.1 M NaOH. Protein concentration was determined by the method of Lowry et al. w27x. 2.9. Statistical analysis Measurements from individual cultures were performed in duplicate and results are expressed as the mean " S.E.M. values for the number of culture preparations indicated. Statistical analysis of the results was determined by oneway analysis of variance followed by the least significant difference multiple range test. In all cases, p - 0.05 was considered significant.
negative correlation Ž r s 0.87. with LDH released ŽFig. 1d.. Glutamate stimulation of cGMP levels was antagonized with NAME Ž1 mM., a potent inhibitor of NOS, and with APV Ž0.1 mM., a competitive antagonist of NMDA glutamate-subtype receptor w17x ŽFig. 2a.. Similarly, either NAME or APV prevented both the increase in LDH release ŽFig. 2b. and the ATP depletion ŽFig. 2c. caused by glutamate. We have previously described that neuronal mitochondrial respiratory chain is damaged following exposure to either ONOOy or PNO donors w6,7x. Accordingly, we were prompted to investigate whether the glutamate-mediated loss of ATP content found was related to the impairment of mitochondrial function. As shown in Table 1, the exposure to glutamate Ž0.1 mM or 1 mM. decreased, by about 20–25%, the activity of succinate cytochrome c reductase, although NADH–CoQ reductase activity remained unchanged in the surviving neurones. The activity of cytochrome c oxidase was significantly decreased Ž31%. when neurones were exposed to 1 mM glutamate, but unchanged by 0.1 mM glutamate. Citrate synthase activity was unaffected by the treatment Žin nmolrminrmg of protein, control, 137.1 " 7.04, n s 4; 1 mM glutamate,
3. Results It is accepted that the increase of cGMP may be regarded as an index of endogenous NO formation w18x. Thus, our results show that a brief Ž15 min. exposure of neurones to glutamate resulted in a dose-dependent increase in cGMP concentration to about 0.05 mM glutamate ŽFig. 1a.. Increasing glutamate concentration further did not result in higher cGMP concentration ŽFig. 1a., possibly reflecting maximal guanylate cyclase activity. In addition, glutamate exposure caused neurotoxicity as shown by the increase of LDH released to the culture medium w23x; the phenomenon was dose-dependent and delayed Ž24 h. ŽFig. 1b.. This effect was accompanied by a decrease in neuronal ATP concentration ŽFig. 1c., showing a significant
Table 2 Effect of glutamate exposure on neuronal respiration Condition
Oxygen consumption Žng atoms Orminrmg. Glucose
Succinate
Control Glu 1 mM Glu 1 mMqNAME Glu 1 mMqAPV
6.61"0.39 4.89"0.32 a 6.11"0.56 5.99"0.53
7.10"0.67 4.35"0.36 a 7.65"0.61 6.68"0.89
Neurones were exposed to 1 mM glutamate for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. Neuronal respiration was measured as described in Section 2 Žsee Fig. 3.. Results are the mean"S.E.M. values from three different culture preparations. a Significantly different when compared to control group.
A. Almeida et al.r Brain Research 790 (1998) 209–216
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gen consumption; 39% of inhibition.. These effects were prevented by NAME or APV ŽTable 2.. A diagrammed run for each condition is shown in Fig. 3. GSH is an important intracellular defence against no mediated mitochondrial damage in neurones w6,7x. In view of this, we have determined GSH concentration in the neurones exposed to glutamate. Fig. 3 shows that a brief Ž15 min. glutamate exposure to the neurones significantly decreased GSH concentration by 20% after 24 h of incubation. This fall in GSH concentration caused by glutamate exposure was prevented by either inhibiting NOS with NAME or by antagonizing NMDA receptor glutamate-subtype with APV ŽFig. 4.. Fig. 3. Oxygen consumption rates in neuronal suspensions under several conditions. Neurones were exposed, or not, to 1 mM glutamate for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. Overall neuronal respiration was measured in cells resuspended in buffered Hanks’ solution with glucose Ž5 mM. as substrate. After inhibition of NADH-linked oxygen consumption with rotenone Ž10 m M., the rate of FADH-linked oxygen consumption Žantimycin-sensitive. was monitored with succinate Ž10 mM. as substrate. Mean slopes values for each condition are shown in Table 2.
135.1 " 8.0, n s 4.. The decrease in succinate cytochrome c reductase and cytochrome c oxidase activities caused by glutamate was completely prevented by NAME or by APV ŽTable 1.. In order to study whether glutamate-mediated inhibition of the respiratory chain components would affect to the respiratory function of the mitochondria, we measured the rate of oxygen consumption by the neurones exposed to glutamate. A brief Ž15 min. glutamate exposure to the neurones decreased, after the 24 h incubation period, the rates of respiration using both glucose Ž26% of inhibition. or succinate plus rotenone Žto inhibit NADH-linked oxy-
Fig. 4. The effect of glutamate exposure on GSH concentration and the protective effects of NAME and APV in neurones in primary culture. Neurones were exposed, or not, to glutamate Ž0.1 or 1 mM. for 15 min either in the absence or in the presence of NAME Ž1 mM. or APV Ž0.1 mM. and the cells were incubated for further 24 h in the absence of effectors. GSH concentration was measured in the cell lysates as described in Section 2. Results are the mean"S.E.M. values from four to five different culture preparations. )Significantly different when compared to control group.
4. Discussion Our results showing the dose-dependent, PNO-mediated increase in cGMP by glutamate exposure ŽFigs. 1 and 2. in neurones confirm those by others w18x and suggest that the neuronal cultures used in the present study may be considered as ‘mature’ and hence appropriate for studies involving glutamate neurotoxicity w15x. In addition, the brief glutamate exposure caused delayed PNO-mediated neurotoxicity, an effect that has been observed both in vitro w15x and in vivo w42x. The dose-dependent decrease in neuronal ATP concentration significantly correlated with cell death ŽFig. 1d., suggesting that impairment of energy metabolism may be a phenomenon related to glutamate-mediated neurotoxicity. This is in agreement with the study of Tsuji et al. w47x, who reported a rapid and irreversible decrease in spinal neuronal ATP by glutamate exposure. However, we have expanded this observation and show that ATP depletion is prevented by NAME ŽFig. 2c., strongly suggesting that neuronal energy impairment leading to neurotoxicity caused by glutamate-receptor stimulation is a process mediated by NOS activity. Several hypotheses involving a possible energy impairment have been postulated to explain the mechanism of glutamate-mediated PNO neurotoxicity. Thus, glutamate receptor stimulation causes activation of polyŽADP ribose. synthetase DNA-repair system, which requires ATP and hence might lead to neuronal energy deficiency w53x. However, blockade of polyŽADP ribose. synthetase activation has never been shown to prevent glutamate receptor-mediated ATP depletion in neurones and, therefore, the relevance of this mechanism for glutamate–NO neurotoxicity remains elusive. Another hypothesis involves the NO-mediated inhibition of the activity of the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase w30,54x. However, it has been shown that in these circumstances the extent of the inhibition is not sufficient to compromise cellular energy production w16,20x. Mitochondria have been considered to be a possible target for glutamate neurotoxicity, because calcium accumulation within this organelle has been observed after
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short exposures to glutamate w50,52x. However, the possible implication of PNO in these phenomena was not investigated. In the present work, we show that a brief glutamate exposure to the neurones caused a significant impairment of mitochondrial succinate–cytochrome c reductase Ž20.6% and 24.4% of inhibition at 0.1 mM and 1 mM glutamate, respectively. and cytochrome c oxidase Ž30.5% of inhibition at 1 mM glutamate. activities, without affecting NADH–CoQ1 reductase activity ŽTable 1.. These effects were completely prevented by APV or by NAME, suggesting that the inhibitory effects were mediated by the pathway involving NMDA-receptor stimulation leading to increased NOS activity. In addition, our results suggest that the inhibition of the respiratory chain by glutamate is irreversible, because it is observed after 24 h of incubation without glutamate and is not reversed by the collection, homogenization and dilution of the samples. Furthermore, this mitochondrial damage caused by glutamate impaired oxygen consumption ŽTable 2. and depleted ATP in the neurones ŽFig. 1c, Fig. 2.. Since the mitochondrial function parameters ŽFig. 1c, Fig. 2c; Tables 1 and 2. were determined only in the surviving neurones, our results strongly suggest that the observed mitochondrial damage preceded neuronal death. Glutamate-receptor stimulation causes a burst of Oy 2 formation w24x, suggesting that the endogenous synthesis of ONOOy under our conditions may be achieved. If so, the resulting ONOOy may be responsible for the direct, irreversible damage to succinate–cytochrome c reductase, which occurs even at very low glutamate concentration ŽTable 1.. This is in agreement with previous observation using authentic ONOOy in either isolated brain mitochondria w6x or submitochondrial particles w26x. Irreversible inhibition of cytochrome c oxidase activity after glutamate exposure ŽTable 1. has been observed in astrocytes w8x after iNOS induction, or in neurones w6x and isolated heart mitochondria w38x after ONOOy exposure. The exact mechanism causing irreversible cytochrome c oxidase inhibition by ONOOy is unknown. However, ONOOy is a well-known lipid pro-oxidant w37x and cardiolipin integrity is absolutely necessary for cytochrome c oxidase activity w45x. Since inhibition of cytochrome c oxidase activity is prevented by the antioxidant trolox w21x, it might be suggested that irreversible damage to cytochrome c oxidase by glutamate exposure could be carried out indirectly, possibly though ONOOy-mediated cardiolipin peroxidation. Finally, the lack of effect of glutamate exposure on neuronal NADH–CoQ reductase activity ŽTable 1. is consistent with the lack of effect of authentic ONOOy on this enzyme activity in neuronal cultures w6x, though other laboratories have observed a direct effect of ONOOy on respiration at NADH-linked substrates in submitochondrial particles w26x. This discrepancy can be explained by the preservation of the antioxidant status in the intact neurone. In fact, glutamate exposure to neurones has been shown to be accompanied by GSH depletion w34x and increased
oxidative stress. Furthermore, under our experimental conditions, GSH depletion by glutamate exposure was prevented by NAME ŽFig. 3., suggesting that NOS activity is involved in the effect. Whether GSH depletion is due to P NO-dependent inhibition of cystine uptake w33x or to a putative reaction of GSH with ONOOy w37x is unknown. In any case, the antioxidant reserve of neurones seems to be compromised under this situation, a fact that may favor the mitochondrial susceptibility to oxidative stress. The pathophysiological relevance of mitochondrial impairment caused by excessive synthesis of PNO in the CNS is well-accepted. Thus, neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, Hungtinton’s diseases or multiple sclerosis are accompanied by the impairment of the mitochondrial function w2,5,35x. Whilst a role for inducible NOS in glial cells have been ascribed in the pathology of these disorders w32x, there is also evidence suggesting that excessive glutamate-receptor stimulation may be involved in the neurodegenerative process w13,28x. Moreover, necrosis and apoptosis are associated with impairment of mitochondrial function. Thus, severe mitochondrial damage has been associated with necrosis, while a remanent mitochondrial function is required for an active apoptosis w1,9,25x. The present results showing that glutamate-receptor stimulation causes an dependent irreversible inhibition of mitochondrial respiration strongly supports the hypothesis that cellular energy deficiency might be a key factor in glutamate neurotoxicity and neurodegeneration. Acknowledgements A.A. is a recipient of an ‘Acciones de Reincorporacion ´ de Doctores y Tecnologos del M.E.C.’, Spain. J.P.B. and ´ S.J.R.H. are in receipt of a Biomedical Collaboration Grant from the Wellcome Trust, U.K. This work was supported by the F.I.S.S.S. and D.G.I.C.Y.T. ŽJ.M.M.. and by the Junta de Castilla y Leon ´ and C.I.C.Y.T. ŽJ.P.B.., Spain. References w1x M. Ankarcrona, J.M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S.A. Lipton, P. Nicotera, Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron 15 Ž1995. 961–973. w2x M.F. Beal, B.T. Hyman, W. Koroshetz, Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases?, Trends Neurosci. 16 Ž1993. 125–131. w3x J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall, B.A. Freeman, Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 1620–1624. w4x K.M. Boje, P.K. Arora, Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death, Brain Res. 587 Ž1992. 250–256. w5x J.P. Bolanos, ˜ A. Almeida, V. Stewart, S. Peuchen, J.M. Land, J.B. Clark, S.J.R. Heales, Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases, J. Neurochem. 68 Ž1997. 2227–2240.
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