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The Journal of Neuroscience, March 15, 2000, 20(6):2094-2103

Exacerbated Responses to Oxidative Stress by an Na⁺ Load in Isolated Nerve Terminals: the Role of ATP Depletion and Rise of [Ca²⁺]_i

Christos Chinopoulos, Laszlo Tretter, Adrienn Rozsa, and Vera Adam-Vizi

Department of Medical Biochemistry, Neurochemical Group, Semmelweis University of Medicine, Budapest H-1444, Hungary

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have explored the consequences of a [Na⁺]_i load and oxidative stress in isolated nerve terminals. The Na⁺ load was achieved by veratridine (5-40 µM), which allows Na⁺ entry via a voltage-operated Na⁺ channel, and oxidative stress was induced by hydrogen peroxide (0.1-0.5 mM). Remarkably, neither the [Na⁺]_i load nor exposure to H₂O₂ had any major effect on [Ca²⁺]_i, mitochondrial membrane potential (m), or ATP level. However, the combination of an Na⁺ load and oxidative stress caused ATP depletion, a collapse of m, and a progressive deregulation of [Ca²⁺]_i and [Na⁺]_i homeostasis. The decrease in the ATP level was unrelated to an increase in [Ca²⁺]_i and paralleled the rise in [Na⁺]_i. The loss of m was prevented in the absence of Ca²⁺ but unaltered in the presence of cyclosporin A. We conclude that the increased ATP consumption by the Na,K-ATPase that results from a modest [Na⁺]_i load places an additional demand on mitochondria metabolically compromised by an oxidative stress, which are unable to produce a sufficient amount of ATP to fuel the ATP-driven ion pumps. This results in a deregulation of [Na⁺]_i and [Ca²⁺]_i, and as a result of the latter, collapse of m. The vicious cycle generated in the combined presence of Na⁺ load and oxidative stress could be an important factor in the neuronal injury produced by ischemia or excitotoxicity, in which the oxidative insult is superimposed on a disturbed Na⁺ homeostasis.

Key words: oxidative stress; Na⁺ load; mitochondrial membrane potential; ATP depletion; Na⁺ deregulation; Ca²⁺ deregulation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Oxidative stress has been associated with neuronal death observed in a variety of neurodegenerative diseases and in ischemia (Schmidley, 1990; Phillis, 1994) (see also Beal, 1995). Hydrogen peroxide is a convenient means to model oxidative stress, because the insult is relatively mild compared with that induced by other reactive oxygen species (Zoccarato et al., 1995; Tretter and Adam-Vizi, 1996), thus enabling the resolution of early alterations in cellular functions. It has been suggested that excessive production of this oxidant contributes to the pathogenesis of Parkinson's disease (Schapira, 1994) and cellular damage occurring during reperfusion (Turrens et al., 1991; Hyslop et al., 1995).

The dysfunctions developing in nerve terminals during acute exposure to the oxidant include depolarization of the plasma membrane, a small increase in resting [Ca²⁺]_i (Tretter and Adam-Vizi, 1996), and a decrease in the ATP level and [ATP]/[ADP] ratio (Zoccarato et al., 1995; Tretter et al., 1997). Although these changes are modest, the oxidant applied at small concentrations (<1 mM) is able to induce delayed cytotoxicity (Whittemore et al., 1995; Desagher et al., 1996; Gardner et al., 1997; Hoyt et al., 1997).

It appears that it could also be acutely more harmful when the oxidative stress is combined with other burdens. An implication for this has been provided by a recent observation that the membrane potential of in situ mitochondria (m) is maintained in the presence of H₂O₂, but when complex I or the F₀F₁-ATPase are also inhibited, themselves without effect on m, mitochondrial membrane potential collapses (Chinopoulos et al., 1999). It has also been reported that H₂O₂ potentiates a decrease in m induced by glutamate excitotoxicity (Scanlon and Reynolds, 1998).

Oxidative stress is a condition that in vivo often occurs concurrently with other disruptions. In this study we specifically examined the energy state, mitochondrial function, and ion homeostasis in nerve terminals during H₂O₂-induced oxidative stress superimposed on a disruption in Na⁺ homeostasis. The importance of this question is indicated by the observations showing that Na⁺ entry is a critical factor in the cellular injury produced by ischemia/reperfusion (Waxman et al., 1994; Weber and Taylor, 1994; Probert et al., 1997; Stys and Lopachin, 1998; Zhang and Lipton, 1999) (see also Urenjak and Obrenovitch, 1996). Furthermore, it has been reported that disruption in [Na⁺]_i homeostasis developing during ischemia is worsened during reperfusion (Rose et al., 1998; Taylor et al., 1999). The mechanism of the exacerbated deregulation of ions is poorly understood. Injury induced by reperfusion is generally thought to be associated with increased production of reactive oxygen species (Cao et al., 1988; Halliwell, 1992; Siesjö et al., 1995).

Glutamate excitotoxicity is another condition involving both increase in [Na⁺]_i and oxidative stress. Excessive stimulation of NMDA receptors leads to an increase in [Na⁺]_i (Kiedrowski et al., 1994a,b) and has also been demonstrated to result in an overproduction of reactive oxygen species (Coyle and Puttfarcken, 1993; Lafon-Cazal et al., 1993; Patel et al., 1996).

Our study, which is the first to address directly the role of Na⁺ load in the acute cellular responses to oxidative stress, might aid in understanding the factors and mechanisms contributing to cellular injury and death in response to an oxidative insult.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of synaptosomes. Isolated nerve terminals (synaptosomes) were prepared from brain cortex of guinea pigs by a method detailed previously (Adam-Vizi and Ligeti, 1984). Synaptosomes obtained from an 0.8 M sucrose gradient were diluted with ice-cold distilled water to a concentration of 0.32 M and then centrifuged at 20,000 × g for 20 min. The pellet was suspended in 0.32 M sucrose (20 mg/ml protein) and kept on ice, and 50 µl aliquots, for further manipulation, were incubated in a standard medium (in mM: 140 NaCl; 3 KCl; 2 MgCl₂; 2 CaCl₂; 10 PIPES, pH 7.38, and 10 glucose) at 37°C.

Determination of m. Membrane potential of in situ mitochondria was determined by 5,5', 6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), a fluorescence probe that accumulates in mitochondria and forms J-aggregates from monomers. It has been demonstrated that both the fluorescence of J-aggregate at 590 nm (Reers et al., 1991) and that of the monomer at 530 nm (DiLisa et al., 1995) reflect m. Synaptosomes suspended in Ca²⁺-free standard medium were loaded with JC-1 (30 µM) for 15 min at 37°C. After sedimentation and washing, synaptosomes were resuspended (8 mg/ml), and for fluorescence measurements, 50 µl aliquots were diluted in 2 ml of standard medium. Fluorescence intensity was determined at 37°C in a PTI (Monmouth Junction, NJ) Deltascan fluorescence spectrophotometer. We have previously shown (Chinopoulos et al., 1999) that H₂O₂ causes a nonspecific decrease in the signal at 595 nm that is unrelated to m, however fluorescence at 535 nm reliably reflects changes in m, therefore we have used only the emission from the monomer recorded at 535 nm in the present study.

Determination of [Na⁺]_i. Synaptosomes were loaded with sodium-binding benzofuran isophthalate (SBFI; 10 µM) by incubation in a standard medium, in which sodium had been iso-osmotically replaced with sucrose and Pluronic acid (0.3%) was added for 60 min at 37°C. As described previously (Deri and Adam-Vizi, 1993) the use of Na⁺-free medium enables the monitoring of the dye accumulation in synaptosomes during the loading period. After sedimentation and washing, the pellet was resuspended (8 mg/ml), and 50 µl aliquots were used in a cuvette containing 1.5 ml of standard medium. The fluorescence of intrasynaptosomally trapped SBFI was measured using 340/380 nm excitation and 510 nm emission wavelengths in a PTI Deltascan fluorescence spectrophotometer at 37°C. A calibration curve to quantify [Na⁺]_i in millimolar concentration was constructed in the presence of 3 µM gramicidin in a medium containing different concentrations of Na⁺, as described previously (Deri and Adam-Vizi, 1993).

Determination of [Ca²⁺]_i. Nerve terminals were loaded with fura-2 by incubation in the standard medium containing 8 µM fura-2 AM at 37°C (4 mg/ml) for 60 min. After sedimentation and washing synaptosomes were resuspended in the standard medium to give an 8 mg/ml protein concentration, and 50 µl aliquots in 2 ml medium were used for determination. Fluorescence intensity was measured in a PTI Deltascan fluorescence spectrophotometer using 340/380 nm excitation and 510 nm emission wavelengths. [Ca²⁺]_i was calculated using the ratio calibration approach described by Grynkiewicz et al. (1985).

ATP and ADP measurement. ATP and ADP levels were determined according to the luciferin-luciferase method as described by Kauppinen and Nicholls (1986) and detailed previously (Tretter et al., 1997). Bioluminescence was detected with an LKB (Turku, Finland) Luminometer 1251. Results are expressed as nanomoles of ATP per milligram of synaptosomal protein and as [ATP]/[ADP] ratio.

Materials. Standard laboratory chemicals were obtained from Sigma (St. Louis, MO). Fura-2 and SBFI were purchased from Calbiochem (San Diego, CA), JC-1 was obtained from Molecular Probes (Eugene, OR).

Statistics. Results are expressed as mean ± SE values. Statistical significance was calculated using a one-way ANOVA followed by Dunnett's test. Differences were considered significant at a level of p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The effect of oxidative stress combined with a Na⁺ load on [Na⁺]_i homeostasis

To investigate [Na⁺]_i homeostasis in nerve terminals, a Na⁺ load was induced by veratridine, which blocks the inactivation of voltage-dependent Na⁺ channels and shifts the activation to more negative membrane potential, thereby causing persistent channel activation (Catterall, 1980). By allowing Na⁺ entry via these channels, veratridine enhances [Na⁺ ]_i (Deri and Adam-Vizi, 1993) and induces depolarization and Ca²⁺ influx in nerve terminals (Adam-Vizi and Ligeti, 1986). Resting [Na⁺]_i in nerve terminals was 12 ± 2.4 mM (n = 36), and Figure 1a indicates that with the addition of 40 µM veratridine [Na⁺]_i started to increase and attained a stable elevated level within a few minutes. [Na⁺]_i at the end of a 20 min incubation period with 40 µM veratridine was 43 ± 3.1 mM (n = 1 4). Oxidative stress induced by H₂O₂ (0.1 mM) alone caused only a slow and relatively small increase in [Na⁺]_i reaching 21 ± 1.3 mM (n = 1 4) over an incubation period of 20 min (Fig. 1a, trace a) (see also Tretter and Adam-Vizi, 1996). When H₂O₂ was applied subsequently to veratridine, a large additional increase in [Na⁺]_i was induced, which was continuous, and no new [Na⁺]_i equilibrium was attained. It is remarkable that a modest initial [Na⁺]_i load was sufficient for the subsequent oxidative stress to induce a large additional increase in [Na⁺]_i (Fig. 1b). When [Na⁺]_i was higher by only a few millimolar concentration (18 ± 2 mM; n = 1 4) at the time of the oxidant application (in the presence of 5 µM veratridine), [Na⁺]_i was greatly enhanced by 0.1 mM oxidant and reached 58 ± 5 mM (n = 1 4) by the end of a 20 min recording period (Fig. 1b), and the higher the initial [Na⁺]_i, the larger was the extent of the oxidant-induced [Na⁺]_i.

View larger version (25K): [in this window] [in a new window]   Figure 1.   [Na+]i measured in nerve terminals loaded with SBFI. a, H2O2 (0.1 mM) was added at 300 sec without previous treatment (trace a) or 200 sec after stimulation with 40 µM veratridine (trace c). Trace b shows the effect of veratridine applied as indicated, without subsequent addition of H2O2. b, Veratridine was added at 100 sec in 5 (trace a), 10 (trace b), 20 (trace c), or 40 µM concentration (trace d), then H2O2 was given in 0.1 mM concentration. Traces are representative of four independent experiments. Basal [Na+]i was 12 ± 2.4 mM (n = 36). Quantitative data of these experiments are included in Figure 3.

It has been reported that under exposure to veratridine mitochondrial respiration is accelerated to produce sufficient amount of ATP for the Na,K-ATPase (Pastuszko et al., 1981), enabling a new [Na⁺]_i equilibrium at an elevated level to be sustained. The question arises whether an additional [Na⁺]_i rise in the presence of the oxidant could be the result of an impaired extrusion of Na⁺ from the cytosol by the Na,K-ATPase. This was examined by the application of tetrodotoxin (TTX; 1 µM) at different time points to block Na⁺ entry via voltage-operated Na channels (Fig. 2). After addition of TTX subsequent to stimulation with veratridine, [Na⁺]_i started to decrease from an elevated level (38 ± 3 mM; n = 6) and returned close to the baseline level (18 ± 2.3 mM; n = 6; measured 5 min after addition of TTX), reflecting the restoration of the normal Na⁺ equilibrium caused by extrusion of Na⁺ by the Na,K-ATPase (Fig. 2, trace a). When TTX was applied together with the oxidant (Fig. 2, trace b), [Na⁺]_i was only slightly decreased immediately after the application of TTX, but then remained at an elevated level (37 ± 3.1 mM; n = 6; measured 5 min after addition of TTX). Likewise, TTX given 200 sec after the oxidative challenge, at an even higher [Na⁺]_i (65 ± 4 mM; n = 6) (Fig. 2, trace c), prevented further increase in [Na⁺]_i, but [Na⁺]_i showed no tendency of returning to the baseline level (64 ± 3.7 mM; n = 6; measured at the end of the 20 min incubation). These results indicate the inability of the Na,K-ATPase to reestablish normal [Na⁺]_i from an elevated level during exposure to an oxidative insult.

View larger version (26K): [in this window] [in a new window]   Figure 2.   The effect of TTX on [Na+]i given after veratridine in the absence or presence of H2O2. Veratridine (40 µM) was applied at 100 sec, then 1 µM TTX (trace a) or TTX + 0.5 mM H2O2 (b) was applied at 300 sec. For trace c, TTX was applied at 500 sec. For trace d, veratridine and H2O2 were added as indicated without TTX. Traces are representative of three independent experiments made in duplicate.

ATP depletion caused by a combined action of H₂O₂-induced oxidative stress and [Na⁺]_i rise: correlation between ATP depletion and deregulation of [Na⁺]_i

Next we wanted to examine whether an insufficient ATP supply could be responsible for the failure of the Na,K-ATPase in the combined presence of oxidative stress and a [Na⁺]_i load.

It has been reported that incubation of synaptosomes with veratridine leads to a decrease in the ATP content attributable to stimulation of the Na,K-ATPase caused by an increase in [Na⁺]_i (Erecinska and Dagani, 1990; Erecinska et al., 1996). We have shown recently that H₂O₂ decreases NADH production in the citric acid cycle, thus limiting the respiratory capacity in nerve terminals (Chinopoulos et al., 1999), and, consistent with this, decreasing the ATP content (Tretter et al., 1997). The possibility emerges from these observations that mitochondria with an impaired respiratory capacity during oxidative stress may not be able to generate a sufficient amount of ATP to fuel the Na,K-ATPase under an increased demand created by a small rise in [Na⁺]_i. Table 1 indicates that indeed there was a drastic fall in the ATP content and [ATP]/[ADP] ratio when nerve terminals were challenged with H₂O₂ during stimulation with veratridine. The control ATP content corresponds to 1.56 mM ATP concentration in the synaptoplasm (calculated with a cytosolic volume of 2.4 µl/mg protein; Adam-Vizi and Ligeti, 1984) being in good agreement with data previously reported for this preparation (Kauppinen and Nicholls, 1986) (see also Erecinska et al., 1996). It should be mentioned that this ATP level and [ATP]/[ADP] ratio is somewhat smaller than those measured in cultured cells (Silver et al., 1997) or different tissues (Erecinska and Wilson, 1982), although great variations can occur in the [ATP]/[ADP] ratio depending on the activity of the tissues (Erecinska and Wilson, 1982). The low ATP level (0.52 ± 0.03 nmol/mg; corresponding to 216 µM in the synaptoplasm), reached 7 min after application of the oxidative insult, was stable, and no further decrease was seen over an incubation for 20 min (data not shown). The observation that veratridine itself induces a ~25% decrease in ATP level, which could be prevented by preincubation with ouabain, agrees with the findings of Erecinska and Dagani (1990). It is important to note that the ATP depletion induced by H₂O₂ and veratridine was independent of extracellular [Ca²⁺] as in the absence of Ca²⁺ a similar, very low level of ATP (0.48 ± 0.04 nmol/mg; n = 4) was measured. It is also demonstrated in Table 1 that ouabain, which could prevent the excessive utilization of ATP by the Na,K-ATPase, significantly attenuated both the ATP loss (1.24 ± 0.04 vs 0.52 ± 0.03 nmol/mg protein) and the decrease in the [ATP]/[ADP] ratio (2.72 ± 0.18 vs 0.87 ± 0.06 nmol/mg) induced by veratridine plus H₂O₂. The restoration of ATP under this condition was significant, but ouabain failed to fully protect ATP to the level seen with H₂O₂ alone. This may be related to the effect of ouabain on the ATP level in the presence of H₂O₂ (1.77 ± 0.09 nmol/mg). The mechanism for this is unclear but may be the result of an altered [Na⁺]_i and [K⁺]_i, which, together with an inhibition of the TCA cycle by H₂O₂ (Chinopoulos et al., 1999), could result in a larger decrease in the ATP level.

View this table: [in this window] [in a new window]   Table 1.   ATP and [ATP]/[ADP] ratio in the presence of H2O2 and veratridine

These results strongly suggest that compromised mitochondria under oxidative stress are unable to balance an increased ATP demand created by the stimulation of the Na⁺ pump caused by an increase in [Na⁺]_i. Therefore, the sustained Na⁺ load, which itself results in a stable [Na⁺]_i rise, when it has an oxidative insult superimposed on it, could produce a vicious cycle in which the initial [Na⁺]_i load, by stimulating the Na,K-ATPase, leads to an ATP depletion which, in turn, restricts extrusion of Na⁺, leading to an additional increase in [Na⁺]_i. Data shown in Figure 3 appear to reinforce this interpretation. The rise in [Na⁺]_i induced by H₂O₂ in veratridine-treated nerve terminals was remarkably parallel with a decrease in the ATP level (Fig. 3). In agreement with a previous report (Erecinska and Dagani, 1990), veratridine itself (5-40 µM) caused only a small change in the ATP level; in fact, significant decrease was only observed in the presence of 40 µM veratridine (Fig. 3b). However, the addition of H₂O₂ (0.1 or 0.5 mM) induced a large decrease in the ATP level and, parallel with this, higher increases in [Na⁺]_i (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Figure 3.   H2O2 induced increase in [Na+]i and decrease in [ATP]. Additions were as described for Figure 1. Veratridine was given in different concentrations without further addition () or followed by treatment with H2O2 at 300 sec in 0.1 mM () or 0.5 mM () concentrations. [Na+]i (a) and ATP level (b) measured at 720 sec in parallel samples are shown as a function of veratridine concentrations. Data are the average of four determinations ± SE. SE is not shown where it is smaller than the symbol. *Significantly different from data obtained with veratridine alone.

Collapse of m in the presence of veratridine and H₂O₂

Given the ATP depletion caused by the combined presence of oxidative stress and a [Na⁺]_i load, we wanted to investigate the state of mitochondria under this condition. For this, m was measured in situ by monitoring the fluorescence of JC-1 at 535 nm. Figure 4, trace b, shows that JC-1 monomer fluorescence was only marginally and transiently increased in the presence of 40 µM veratridine, indicating that plasma membrane depolarization and an increase in [Na⁺]_i have no significant influence on m. Application of veratridine in higher concentrations (up to 80 µM) gave essentially the same result (data not shown). We have reported recently that H₂O₂ itself has no effect on m (Chinopoulos et al., 1999), which is also demonstrated in Figure 4, trace a. However, when H₂O₂ (0.1-0.5 mM) was applied to nerve terminals depolarized previously by veratridine (40 µM), an increase in the monomer fluorescence was observed that was proportional to the concentrations of H₂O₂ (Fig. 4, traces c-e). A concentration of 0.5 mM H₂O₂ applied after veratridine nearly completely collapsed m over an incubation period of 20 min. The effect of H₂O₂ on m was dependent on Ca²⁺; in the absence of extracellular Ca²⁺ (no added Ca²⁺ + 100 µM EGTA present in the medium), addition of H₂O₂ subsequent to veratridine produced a significantly attenuated change in m (Fig. 5), suggesting that the effect of H₂O₂ on m was associated with a rise in [Ca²⁺]_i. Pretreatment of synaptosomes with 10 µM cyclosporin A had no influence on the collapse of m induced by veratridine and H₂O₂ (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 4.   Fluorescence of JC-1 at 535 nm in the presence of veratridine and H2O2. Synaptosomes loaded with JC-1 were incubated in a standard medium (0.2 mg/ml). Veratridine (40 µM) was added at 100 sec followed by addition of H2O2 at 300 sec in 0.1 mM (trace c), 0.2 mM (trace d), or 0.5 mM concentrations (trace e). Traces a and b show the effects of 0.5 mM H2O2 (a) and 40 µM veratridine (b), respectively, given at 300 sec. Traces are representative of four independent experiments. A 1 µM concentration of FCCP was added at the end of each experiment to generate a signal representing the total collapse of m. Quantitative data of these experiments are included in Table 2.

View larger version (18K): [in this window] [in a new window]   Figure 5.   The combined effect of veratridine and H2O2 on m is dependent on the presence of Ca2+ in the medium. JC-1 fluorescence at 535 nm in response to veratridine (40 µM) and H2O2 (0.5 mM) was measured in synaptosomes in the presence of 2 mM Ca2+ (trace c) or when Ca2+ was lacking in the medium (no Ca2+ was added, and 100 µM EGTA was present) (trace b). Trace a shows the effect of 0.5 mM H2O2 (300 sec) added subsequent to 40 mM K+ (100 sec). Traces are representative of three experiments. Quantitative data of these experiments are included in Table 2.

We addressed the question whether depolarization of the plasma membrane, or alternatively an increase in [Na⁺]_i induced by veratridine, plays a role in the loss of m by H₂O₂ when added after veratridine. To resolve this, we applied an alternative means to depolarize nerve terminals, using high [K⁺], which activates voltage-operated calcium channels (VOCCs), giving rise to a [Ca²⁺]_i signal (Ashley et al., 1984) without inducing any change in [Na⁺]_i (Deri and Adam-Vizi, 1993). Figure 5 shows that 40 mM [K⁺] itself did not influence m, and H₂O₂ (0.5 mM) added 200 sec after K⁺ had only a marginal effect. This is in marked contrast to what was observed when H₂O₂ was added after veratridine, in spite of a larger depolarization induced by 40 mM K⁺ (~43 mV) than that caused by 40 µM veratridine (~28 mV; Adam-Vizi and Ligeti, 1984). These results suggest that plasma membrane depolarization, even when sustained for 20 min, has no influence on m; in contrast, an increase in [Na⁺]_i appears to have a great impact on the state of mitochondria subsequently exposed to an oxidative insult. It is important to note that increase in [Na⁺]_i itself, in the absence of oxidative stress, has no effect on m; even very high [Na⁺]_i alone, in the presence of 500 µM ouabain and 40 µM veratridine (~70-80 mM), was without significant effect on m (data not shown).

A statistical summary of the results shown in Figures 4 and 5 is given in Table 2, indicating that H₂O₂ in combination with veratridine significantly increased the fluorescence of JC-1 at 535 nm. A large part of this required the presence of Ca²⁺ in the medium; in the absence of Ca²⁺ the increase in the fluorescence, although statistically significant, was marginal.

View this table: [in this window] [in a new window]   Table 2.   Statistical analysis of data on fluorescence of JC-1 at 535 nm in the presence of veratridine and H2O2

[Ca²⁺]_i rise in the presence of veratridine and H₂O₂ is parallel with a decrease in m

The question arises why m collapses under oxidative stress when combined with a [Na⁺]_i load and what is reflected in the Ca²⁺-dependent character of the loss of m.

To determine whether oxidative stress could enhance the veratridine-evoked [Ca²⁺]_i increase accounting for the collapse of m, [Ca²⁺]_i was measured under identical conditions to those used in the experiments for monitoring m. We found (Fig. 6, Table 3) that veratridine (40 µM) induced a moderate rise in [Ca²⁺]_i and, similarly, H₂O₂ itself caused only a slow and small increase in [Ca²⁺]_i (White and Clarke, 1988) (see also Tretter and Adam-Vizi 1996). However, after addition of H₂O₂ (0.1-0.5 mM) 200 sec after stimulation with veratridine, [Ca²⁺]_i started to increase further, and by the end of an incubation for 20 min [Ca²⁺]_i reached 2100 ± 61 nM in the presence of 0.5 mM H₂O₂ (Fig. 6, traces e-g, Table 3). This is likely to be an underestimated value, given the low K_m of fura-2 for Ca²⁺ (Hyrc et al., 1997). The rise in [Ca²⁺]_i induced by H₂O₂ and [Na⁺]_i load was unaltered by pretreatment with 10 µM cyclosporin A (data not shown). The rate of change of [Ca²⁺]_i in the presence of veratridine and H₂O₂ is very similar to that of m shown in Figure 4. This, and the Ca²⁺ dependency of the decrease in m by H₂O₂ shown in Figure 5, suggest that depolarization of mitochondria is related to an enhanced [Ca²⁺]_i rise induced by the oxidant in Na⁺-loaded nerve terminals. Consistent with this, H₂O₂ applied 200 sec after plasma membrane depolarization by 40 mM K⁺, a condition resulting in no change of m (Fig. 5, trace a), failed to induce a significant increase in [Ca²⁺]_i (Fig. 6).

View larger version (26K): [in this window] [in a new window]   Figure 6.   [Ca2+]i measured in synaptosomes loaded with fura-2. Nerve terminals were depolarized by 40 mM K+ (traces b-d) or 40 µM veratridine (traces e-g) applied at 100 sec, then H2O2 was added in 0.1, 0.2, or 0.5 mM concentrations as indicated. Trace a shows the effect of 0.5 mM H2O2 given at 100 sec. Traces are representative of four determinations.

View this table: [in this window] [in a new window]   Table 3.   [Ca2+]i 5 or 15 min after addition of H2O2 in the presence of veratridine

The correlation between an enhanced [Ca²⁺]_i rise and a fall in m was reinforced by the effect of TTX. Addition of TTX (1 µM) to inhibit voltage-dependent Na⁺ channels 200 sec after imposition of the oxidative stress (Fig. 7) significantly attenuated the H₂O₂-induced [Ca²⁺]_i rise; [Ca²⁺]_i increased from 820 ± 30 to 1110 ± 50 nM (n = 3) during the incubation period with TTX, whereas over a same period of incubation without TTX [Ca²⁺]_i reached 1910 ± 70 nM (n = 3). Parallel with changes in [Ca²⁺]_i, the decrease in m induced by H₂O₂ was also attenuated by TTX (Fig. 7b). This also indicates that Na⁺ entry is a critical factor both in the large increase of [Ca²⁺]_i and in the mitochondrial depolarization occurring in the combined presence of veratridine and H₂O₂. These results suggest that the collapse of m is very likely to result from a large increase in [Ca²⁺]_i occurring when oxidative stress is superimposed on a [Na⁺]_i load.

View larger version (22K): [in this window] [in a new window]   Figure 7.   [Ca2+]i rise and depolarization of m induced by H2O2 (0.5 mM) and veratridine (40 µM) is diminished after addition of TTX. Veratridine and H2O2 were added as indicated, then 200 sec after H2O2, TTX (1 µM) was applied, and [Ca2+]i (a) and JC-1 fluorescence (b) were measured in parallel samples loaded with fura-2 or JC-1. Traces are representative of three independent experiments.

Basis for increase in [Ca²⁺]_i

Figure 6 and Table 3 indicate that oxidative stress and [Na⁺]_i load initiate a large increase in Ca²⁺, which does not attain a new equilibrium, but rather exhibits the tendency of a continuous, uncontrolled [Ca²⁺]_i rise. The question arises as to what the underlying mechanism for this apparent Ca²⁺ deregulation could be.

Entry via VOCCs

The [Ca²⁺]_i signal after depolarization by high [K⁺] is the result of activation of VOCCs followed by a rapid inactivation (Ashley et al., 1984; Alvarez Maubecin et al., 1995), thus the lack of effect of H₂O₂ on [Ca²⁺]_i applied after K depolarization shows that H₂O₂ has no effect on VOCCs under these conditions. This was also indicated by the results that both [Ca²⁺]_i rise and mitochondrial depolarization elicited by H₂O₂ in Na⁺-loaded synaptosomes were unaltered by pretreatment with inhibitors of N-, P-, Q- or L-type Ca²⁺ channels (-conotoxin, 1 µM; -agatoxin IVA, 50 nM; -conotoxin MVIIC, 1 µM; and tai-conotoxin, 160 nM, respectively; n = 3; data not shown). When nerve terminals incubated in Ca²⁺-free medium were challenged with veratridine plus the oxidant, no change in [Ca²⁺]_i was produced (data not shown). These results indicate that extracellular Ca²⁺ is involved in the oxidant-induced [Ca²⁺]_i rise, but no Ca²⁺ entry is likely to be mediated by VOCCs.

This latter finding requires a comment, because it has been shown recently that [Ca²⁺]_i signal is enhanced when high [K⁺] is applied in the presence of the oxidant (Tretter et al., 1997), and consistent with this, H₂O₂ has been reported to enhance Ca²⁺ influx via VOCCs (Li et al., 1998). The lack of effect of the oxidant on [Ca²⁺]_i applied after the depolarizing stimulus in this study might indicate that oxidative conditions should be present at the onset of the activation of VOCCs for the Ca²⁺ influx to be enhanced, and addition of H₂O₂ after the stimulus, even during a sustained depolarization, is no longer able to influence Ca²⁺ influx.

Activation of glutamate receptors

We also considered whether glutamate, which is assumed to be released from nerve terminals when stimulated with veratridine and H₂O₂, could contribute to the increase in [Ca²⁺]_i, but neither the NMDA receptor antagonist MK 801 (10 µM) nor GYKI 52466 (50 µM), blocker of the AMPA receptors had any influence on the [Ca²⁺]_i rise induced by 40 µM veratridine and 0.5 mM H₂O₂ (data not shown).

Effect of mitochondrial depolarization

Collapse of m in many cells gives rise to an elevated [Ca²⁺]_i owing to an impaired buffering of Ca²⁺ by mitochondria (Budd and Nicholls, 1996; Wang and Thayer, 1996; White and Reynolds, 1996). In nerve terminals no change in [Ca²⁺]_i could be observed after dissipation of m by rotenone (2 µM)/oligomycin (10 µM) either in the presence or in the absence of extracellular Ca²⁺ (data not shown), thus it is unlikely that [Ca²⁺]_i rise induced by veratridine and H₂O₂ could be secondary to a loss of m.

Inhibition of plasmalemmal Ca²⁺-ATPase

Given the severe ATP depletion induced by H₂O₂ in the presence of veratridine, it is possible to propose that the large increase in [Ca²⁺]_i under this condition results from an impaired ATP-dependent Ca²⁺ removal, primarily by the plasmalemmal Ca²⁺-ATPase. This prediction was supported by experiments with ouabain, which is able to preserve a significant part of ATP during stimulation with veratridine and H₂O₂ (Table 1). Figure 8 shows that in the simultaneous presence of ouabain (500 µM) and veratridine (40 µM), [Ca²⁺]_i increased to a slightly higher level compared to that observed with veratridine alone. This is in agreement with a higher [Na⁺]_i possibly driving more Ca²⁺ into the terminals via the Na⁺-Ca²⁺ exchanger known to be present in the plasma membrane of nerve terminals (Gill, 1982; Sanchez-Armass and Blaustein, 1987). The presence of ouabain along with veratridine attenuated the H₂O₂-induced increase in [Ca²⁺]_i (Fig. 8). The data in Table 4 show that ouabain attenuated [Ca²⁺]_i rise induced by H₂O₂ in Na⁺-loaded synaptosomes by ~50%. The remarkable correlation between the fall in [ATP] and the rise in [Ca²⁺]_i suggests that inhibition of the ATP-dependent removal of [Ca²⁺]_i by the Ca²⁺-ATPase in the plasmalemma is the cause of the increased [Ca²⁺]_i. The evidence argues against the involvement of Ca²⁺ entry via reversal of the plasmalemma Na⁺-Ca²⁺ exchanger. Although 40 µM veratridine + 500 µM ouabain increased [Na⁺]_i to 70-80 mM within a few minutes (data not shown), the Ca²⁺ rise was only slightly higher than that caused by veratridine alone, where [Na⁺]_i rose only to 40 mM (Fig. 8). However, even this result does not entirely rule out the possibility that when the ATP level is also reduced simultaneously, as observed with veratridine plus H₂O₂ (but not with veratridine plus ouabain), the reverse function of the Na⁺-Ca²⁺ exchanger could become significant. Unfortunately experiments with inhibitors of the Na⁺-Ca²⁺ exchanger (Bepridil, 10 µM; 3',4'-dichlorobenzamil; 10 µM) gave ambiguous results as Bepridil appears to interfere with Na⁺ channels and prevent the effect of veratridine on [Na⁺]_i, and 3',4'-dichlorobenzamil gives fluorescent signals at the wavelengths used for measuring [Ca²⁺]_i and [Na⁺]_i (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 8.   [Ca2+]i rise induced by veratridine and H2O2 in the presence or absence of ouabain. Veratridine (40 µM) and H2O2 (0.2 mM) were added as indicated (a), and [Ca2+]i was measured in fura-2-loaded nerve terminals. Ouabain (500 µM) was given 50 sec before veratridine (b), as indicated by the arrow. Traces are representative of three experiments. Quantitative data are given in Table 4.

View this table: [in this window] [in a new window]   Table 4.   Comparison of the effect of H2O2 on [Ca2+]i in the presence of veratridine with or without ouabain

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The major observation in the present study is that when oxidative stress occurs together with a Na⁺ load, the damaging effect of oxidative stress is greatly exacerbated. A key element in the dysfunction is a large fall in [ATP] in the combined presence of H₂O₂ and Na⁺ load. The basis for this is an increased utilization of ATP by the Na⁺ pump activated by a Na⁺ entry coupled with the inability of mitochondria to respond adequately with increasing ATP production because of limitation of the respiratory capacity by H₂O₂. This leads to a vicious cycle in which the [Na⁺]_i increase augments the decrease in [ATP], which, in turn, further inhibits the Na⁺ pump, enhancing the [Na⁺]_i increase. The large fall in ATP gives rise to the inability to remove cytosolic Ca²⁺ via the plasmalemmal ATPase. The resulting Ca²⁺ accumulation then leads to a collapse of m.

A crucial effect of H₂O₂ in the early stage of the oxidative insult appears to be the inhibition of -ketoglutarate dehydrogenase and, as a consequence, a decrease in the mitochondrial NADH production (Chinopoulos et al., 1999). The ATP level under this condition, although decreased (Tretter et al., 1997), is still adequate to secure a resting function of the ATP-driven ion pumps in the plasma membrane, thus, the Na⁺ and Ca²⁺ electrochemical gradients are only slightly decreased (Tretter and Adam-Vizi, 1996), and m is maintained (Chinopoulos et al., 1999).

When oxidative stress is imposed on nerve terminals in which [Na⁺]_i is increased, a complex dysfunction develops with (1) a drastic fall in the ATP level, (2) a deregulation of [Na⁺]_i and [Ca²⁺]_i, and (3) the loss of m. In the interpretation of these observations, the following questions have to be addressed: (1) what are the underlying mechanisms and the sequence of these changes?, and (2) what is their relevance to pathological conditions in which oxidative stress is assumed to have a pivotal role?

Fall in the ATP level

H₂O₂ when given alone, produces not more than ~30% decrease in the ATP level after incubation for 7 min (Table 1; Tretter et al., 1997). However, owing to a combined effect of oxidative stress and Na⁺ load, the energy resources of nerve terminals are almost completely drained (Table 1, Fig. 3). The most obvious explanation for the development of the severe energy deficit is that mitochondria working with a limited respiratory capacity under oxidative stress (Chinopoulos et al., 1999) are unable to produce sufficient amount of ATP when an additional demand presents itself because of stimulation of the Na,K-ATPase by an increased [Na⁺]_i. Preliminary experiments using different mitochondrial inhibitors together with a Na⁺ load appear to be consistent with this interpretation (data not shown).

It is important to note that the energy deficit brought about by H₂O₂ when applied at an elevated [Na⁺]_i is unrelated to an increase in [Ca²⁺]_i (Fig. 6, Table 1). This strongly suggests that the decrease in ATP level is upstream from the large increase in [Ca²⁺]_i. Since, in the absence of Ca²⁺, oxidative stress together with a Na⁺ load deplete ATP at a sustained m, the loss of ATP should be also upstream from the collapse of m.

Dissipation of [Na⁺] and [Ca²⁺] gradients

H₂O₂, when applied after veratridine, at an elevated [Na⁺]_i, induced a large additional increase in [Na⁺]_i (Fig. 1a,b). It appears most likely that, owing to the initial rise in [Na⁺]_i produced by veratridine, the Na,K-ATPase is already stimulated at the onset of the application of oxidative stress, but as a result of the effect of the oxidant on the mitochondria, as discussed above, ATP production becomes insufficient. This, in turn, would limit the function of the Na,K-ATPase, resulting in an additional gradual rise in [Na⁺]_i. At an ATP level of 0.52 ± 0.03 nmol/mg (216 µM) and with an [ATP]/[ADP] ratio reduced to 10% of the control (Table 1), Na⁺ extrusion by the Na,K-ATPase should be severely impaired (K_m for ATP is 200-400 µM; Erecinska and Dagani, 1990), accounting for the gradual collapse of the [Na⁺] gradient. Therefore the picture of a vicious cycle emerges, in which a relatively small Na⁺ load aggravates the effect of oxidative stress creating gradually an energy deficit, which in turn leads to [Na⁺]_i deregulation.

To this picture, another element, a large increase in [Ca²⁺]_i induced by the combination of oxidative stress and [Na⁺]_i load should also be added (Fig. 6, Table 3). The increase is not caused by activation of VOCCs or glutamate receptors because antagonists to these pathways did not affect [Ca²⁺] rise (data not shown).

It has been suggested that Ca²⁺ entry into rat optic nerves during anoxia is mediated by a reverse Na⁺-Ca²⁺ exchange (Stys et al., 1992). This seems unlikely to be the mechanism for the [Ca²⁺]_i increase in this study because when [Na⁺]_i was increased to 70-80 mM by the combination of ouabain and veratridine there was only a very small rise in [Ca²⁺]_i. A caveat here is that the large fall in [ATP] when H₂O₂ and veratridine are used may in some way activate Ca²⁺ entry via this pathway. Such an effect of ATP has been described for the Na⁺-H⁺ exchanger, which has a decreased affinity to H⁺ when the level of ATP is decreased (Orlowski and Grinstein, 1997).

A possible interpretation consistent with our observations is that [Ca²⁺]_i deregulation is related to the ATP depletion evolving from the combined effects of oxidative stress and Na⁺ load. It is expected that, similarly to that of the Na,K-ATPase, the function of the Ca²⁺-ATPase in the plasma membrane becomes also severely limited because of an insufficient ATP supply. The slow pattern of the [Ca²⁺]_i rise (Fig. 6) is consistent with a [Ca²⁺]_i deregulation caused by an impaired Ca²⁺ extrusion by the Ca²⁺-ATPase. This interpretation is reinforced by the result that ouabain, which partly prevents the loss of ATP (Table 1), significantly attenuates the [Ca²⁺]_i rise under this condition (Fig. 8).

Loss of m

The gradual depolarization of mitochondria in response to H₂O₂ in Na⁺-loaded nerve terminals is clearly a Ca²⁺-dependent process (Fig. 5) and occurs parallel with increases in [Ca²⁺]_i (Fig. 7). It could be expected that when [Ca²⁺]_i is in the micromolar range, the mitochondrial permeability transition would be induced (Duchen et al., 1993), but we obtained no evidence for the involvement of a cyclosporin A-sensitive permeability transition in the collapse of m. This is in contrast with the glutamate-induced mitochondrial depolarization, which is sensitive to cyclosporin A (Schinder et al., 1996; White and Reynolds, 1996). Because m is the driving force for Ca²⁺ uptake by mitochondria, Ca²⁺ uptake itself could discharge m if not balanced by H⁺ extrusion (Nicholls, 1985). This is the most likely mechanism for the Ca²⁺-dependent loss of m observed in the present study, given the limited capacity of the respiratory chain to maintain m in the presence of the oxidant (Chinopoulos et al., 1999).

The increased [Na⁺]_i might also potentiate the effect of Ca²⁺ on mitochondria by accelerating Ca²⁺ efflux via the Na⁺-Ca²⁺ exchanger present in the mitochondria of excitable cells (Crompton et al., 1978), contributing to a futile Ca²⁺ cycling. In addition, reestablishing the Na⁺ gradient across the mitochondrial inner membrane by the mitochondrial Na⁺-H⁺ exchange against a large [Na⁺]_i could also be a contributing factor in the collapse of m.

Relevance to pathological conditions

A small Na⁺ load appears to be sufficient to exacerbate the condition created by oxidative stress, which could be an important contributing factor in the dysfunction developing during excessive stimulation of NMDA receptors or during reperfusion after an anoxic period, when the oxidative insult is superimposed on a disturbed [Na⁺] homeostasis.

It has been demonstrated that in NMDA-stimulated cells (1) [Na⁺]_i is increased (Kiedrowski et al., 1994a, b), (2) m is lost (Budd and Nicholls, 1996; Isaev et al., 1996; Schinder et al., 1996; White and Reynolds, 1996), and (3) reactive oxygen species are produced (Lafon-Cazal et al., 1993; Dugan et al., 1995; Reynolds and Hastings, 1995; Patel et al., 1996). In the light of our observations presented here, it could be assumed that the cytoplasmic Na⁺ elevation, in addition to hampering Ca²⁺ extrusion via the Na⁺-Ca²⁺ exchanger (Kiedrowski et al., 1994a), could contribute to cell death by aggravating the damage caused by the oxidative component of the excitotoxic stimulus. Consistent with this could be a recent report by Scanlon and Reynolds (1998) that exposure of forebrain neurons to hydrogen peroxide potentiated the mitochondrial depolarization caused by glutamate and that by Strijbos et al. (1996) suggesting that a TTX-sensitive Na⁺ entry is part of a vicious cycle that leads to neurodegeneration after stimulation of NMDA receptors.

It is well documented that oxygen-glucose deprivation induces [Na⁺]_i deregulation (Hansen, 1985; Stys et al., 1992; Waxman et al., 1994; for review, see Urenjak and Obrenovitch, 1996), which further worsens during reperfusion (Rose et al., 1998; Taylor et al., 1999). Reperfusion injury is generally thought to be associated with an increased production of reactive oxygen species (Cao et al., 1988; Halliwell, 1992), and consistent with this, presence of H₂O₂ at a concentration of 0.1 mM has been demonstrated in the striatum during reperfusion (Hyslop et al., 1995). The severe energy deficit, the complex Na⁺ and Ca²⁺ deregulation, and the loss of m induced by H₂O₂ in Na⁺-loaded nerve terminals demonstrated in this study could indicate a mechanism by which cellular injury initiated during ischemia could be further augmented during reperfusion, preventing the restoration of normal cellular functions.

	FOOTNOTES

Received July 14, 1999; revised Dec. 21, 1999; accepted Dec. 27, 1999.

This work was supported by grants to V. A.-V. from Orszagos Tudomanyos Kutatasi Alap, Egeszsegugyi Tudomanyos Tanacs, Oktatasi Miniszterium, and Magyar Tudomanyos Akademia. We thank Dr. Michael Duchen for helpful suggestions during preparation of this manuscript. Thanks are expressed to K. Takács and K. Zölde for excellent technical assistance.

Correspondence should be addressed to Dr. Vera Adam-Vizi, Department of Medical Biochemistry, Semmelweis University of Medicine, Budapest, H-1444, P.O. Box 262, Hungary. E-mail: AV{at}puskin.sote.hu.

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