Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

Abstract

It has been reported that the activity of mitochondrial aconitase (m-aconitase) is rapidly inhibited in a variety of cells when exposed to nitric oxide (NO). In present study, we found that NO significantly increased the number of surviving neurons via enhanced mitochondrial functions with simultaneous addition of the [Fe(II)(β-citryl-L-glutamate; β-CG)] complex. In vitro, a variety of aconitase-inhibitors, such as fluorocitrate, cyanide ion, ferricyanide ([Fe(CN)₆]), and various oxidants including superoxide anion, inhibited the activity of m-aconitase even in the presence of Fe(II), whereas a NO-donor, nitroprusside (SNP) ([Fe(CN)₅NO]), was the only agent that significantly increased activity of that enzyme. Therefore, it is reasonable to assume that NO released from SNP promotes Fe-dependent activation of aconitase. All other tested NO-donors, including 3-morpholino-sydnonimine (SIN), Deta NONOate (NOC18), and NaNO₂, also promoted activation of m-aconitase in time- and dose-dependent manners in the presence of Fe(II). The promoting effects of the NO-donors on activation disappeared with the addition of NO-scavengers. In intact mitochondria, all tested NO-donors promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II), whereas that was not seen in its absence. These findings suggest that NO released from NO-donors promotes Fe-dependent activation of aconitase. In mixed neuronal and glial cultures, NO-donors except for SNP enhanced mitochondrial activity at low concentrations. Furthermore, simultaneous addition of the [Fe(II)(β-CG)] complex significantly enhanced those activities and greatly increased the number of surviving neurons. Thus, NO can carry Fe ions into m-aconitase via the guide of the tag of β-CG addressed to the enzyme.

Aconitase is a critical citric acid-cycle enzyme that catalyzes the stereospecific interconversion of citrate and isocitrate via the intermediate cis-aconitate, whose activities depend on an intact [4Fe–4S]²⁺ cluster.¹⁾ This enzyme contains a unique [4Fe–4S]²⁺ cubane cluster in its active catalytic sites, with one particularly labile Fe atom (so-called Fe_a). Loss of aconitase activity is commonly used as a biomarker of oxidative damage due to susceptibility of the [4Fe–4S]²⁺ cubane cluster to oxidative disassembly.²⁾ Oxidatively inactivated aconitase is rapidly reactivated in vitro and in vivo, by cluster reduction and Fe(II) ion reinsertion.³⁾ However, the physiological mechanisms for reduction of [3Fe–4S]⁺ and Fe(II) insertion into the [3Fe–4S]⁰ center are currently unknown.²⁾ In cells, aconitase is in a dynamic state of inactivation and reactivation, while the Fe–S center is under continuous threat from attack by physiological oxidants including superoxide anion, hydrogen peroxide (H₂O₂), molecular oxygen, and nitric oxide (NO), and perhaps even peroxynitrite (ONOO⁻). Significant inactivation of aconitase has been reported to occur after exposure to such oxidants.²⁾

NO is a free radical produced by a wide variety of cell types, that reacts predominantly with Fe(III) or Fe(II) ions of heme proteins and Fe–S centers, as well as molecular oxygen, and superoxide anion.^4,5) Moreover, NO easily reaches mitochondria from cytosolic or extracellular sources, due to its low molecular radius and hydrophobic nature.⁶⁾ It has also been reported that induction of NO synthesis or exposure of different cell types to NO-donors resulted in early loss of mitochondrial aconitase (m-aconitase) activity.¹⁾

NO-mediated inactivation of m-aconitase has been reported in a variety of cells including macrophages,⁷⁾ fibroblasts,⁸⁾ tumor cells,^9,10) and Escherichia (E.) coli,¹¹⁾ though results from in vitro studies are somewhat contradictory. Indeed, in vitro studies that used porcine heart m-aconitase found that low concentrations of NO did not inactivate aconitase, whereas high concentrations led to moderate inhibition.¹²⁾ Resistance of purified E. coli aconitase and human recombinant cytosolic aconitase (c-aconitase) to NO-dependent inactivation has also been reported.¹³⁾ On the contrary, inactivation by either NO or a NO-donor was reported for both m-aconitase and c-aconitase in the presence and absence of a substrate.¹⁴⁾ In agreement with those findings, it was shown that the active form [4Fe–4S] of m-aconitase is rapidly and directly oxidized by ONOO⁻, to [3Fe–4S], causing a loss of catalytic activity.¹²⁾ Another report¹¹⁾ also noted that E. coli aconitase is highly sensitive to NO-mediated inactivation apparently independent of ONOO⁻ formation. Recently, it was also reported that NO binds to Fe_a in the [4Fe–4S] cluster of recombinant porcine m-aconitase and slowly promotes complete cluster disassembly.⁶⁾

The present findings demonstrate that NO-donors promote Fe-dependent activation of m-aconitase ([3Fe–4S] form) in vitro. In intact mitochondria, the NO-donors also promoted Fe-dependent reactivation of the enzyme disassembled by ammonium peroxodisulfate (APS). In mixed neuronal and glial cultures, NO-donors except for sodium nitroprusside (SNP) enhanced mitochondrial activity at low concentrations. Furthermore, simultaneous addition of the [Fe(II)(β-citryl-L-glutamate; β-CG)] complex significantly enhanced mitochondrial activities and greatly increased the number of surviving neurons. Thus, NO plays a role as an Fe-carrier to mitochondrial aconitase, and then activates it, while β-CG is considered to function as a tag addressed to the enzyme.

Materials and Methods

Materials

Aconitase (from porcine heart), isocitrate dehydrogenase recombinant (from yeast), xanthine oxidase (from buttermilk), and β-nicotinamide adenine dinucleotide phosphate (NADP⁺) were purchased from Oriental Yeast Co. (Tokyo Japan). Deferoxamine mesylate (Def: Fe(III) chelator), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and DL-fluorocitric acid tribarium were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Amino-3-morpholinyl-1,2,3-oxazolium chloride (SIN chloride), came from Tocris Cookson Inc. (Missouri, U.S.A.). Fe(II)O (Alfa Aesar), Fe(III)₂O₃, ammonium Fe(III)citrate, Fe(II)gluconate (Alfa Aesar), and haemin chloride (Alfa Aesar), came from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), while 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazine (Deta NONOate : NOC18) came from Calbiochem (CA, U.S.A.). Hypoxanthine sodium, ammonium Fe(II) sulfate hexahydrate (Fe(II)AS), nitroprusside sodium (SNP), Fe(II)lactate, and ammonium peroxodisulfate (APS) were obtained from Nacalai Tesque (Kyoto, Japan), while 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO) came from Dojindo (Tokyo Japan). An MTT assay kit (cell proliferation assay kit I) was obtained from Roche (Mannheim, Germany). β-Citryl-L-glutamate (β-CG) was synthesized as previously described.¹⁵⁾ [Fe(II)(Citrate)] complex were prepared from Fe(II)O and trisodium citrate as previously described.¹⁶⁾ All other reagents were purchased from commercial sources at the highest grade available.

Determination of Aconitase Activity

Enzyme activities were assayed by determining the formation of cis-aconitate from isocitrate.¹⁾ UV assays were performed at room temperature. The final volume was 1 mL, including 25 mM Tris–HCl, pH 7.4, and 2 mM trisodium DL-isocitrate. After addition of the enzyme, changes in absorption at 240 nm were determined from 0.5 to 1.5 min. One unit was defined as the amount of enzyme necessary to produce 1 µmol of cis-aconitate/minute (ε_240 nm=3.6 mM⁻¹ cm⁻¹).³⁾ Unless otherwise noted, all specific activities refer to the UV assay. The presence of Fe(II) and a reducing agent, such as cysteine or dithiothreitol, is required for activation of aconitase.³⁾ In some experiments, aconitase activity was determined using a coupled assay,¹⁾ with which NADP reduction was examined.

Activation of Aconitase

Aconitase used in the present study is inactive ([3Fe–4S] cluster form), and requires activation ([4Fe–4S] cluster form) by Fe(II) and a reducing agent.³⁾ Aconitase was activated in a routine manner by adding a solution composed of ferrous ammonium sulfate (Fe(II)AS: 5–100 µM) at 37°C for 10 min to small tubes containing 50 mM Tris–HCl buffer, pH 7.4, and 2.5 mM dithiothreitol in a total volume of 50–100 µL. In some experiments, the activated aconitase protein solution was rapidly desalted using a Bio-Gel p-6 cartridge (1.6×4 cm) equilibrated with 50 mM Tris–HCl buffer, pH 7.4, containing 0.5 mM dithiothreitol to remove excess low molecular activating agents. These aconitase preparations were used as “activated aconitase” in the experiments.

Treatments of Intact Mitochondria with Oxidants: Effects of NO-Donor on Reactivation of Mitochondrial Aconitase

Mitochondrial fractions were prepared from Wistar rat hearts as previously described¹⁷⁾ and stored at −80°C until use in the experiments.

Mitochondria were diluted to about 25 mg of protein/mL in 100 mM Tris–HCl buffer, pH 7.4, and then incubated at 10 min after addition of 100 µM ammonium peroxodisulfate (APS), a relatively mild oxidant, as previously described.³⁾ Aconitase ([3Fe–4S] cluster form) damaged by APS loses its enzyme activities, while it can be reactivated with Fe(II) ion. All incubations were performed at 37°C. After incubation, 30-µL aliquots of the reaction mixtures were suspended in 30 µL of 1% Triton X-100, and solubilized, then 50-µL aliquots were used for determination of aconitase activity.¹⁶⁾

Primary Cell Cultures from Newborn Mouse Brain Tissues

Cerebrums of 1-d-old ddY mice were dissected and placed in Ca²⁺/Mg²⁺-free Dulbecco’s phosphate-buffered saline [PBS(−)]. Dissociation and cultures of neurons were performed as previously described,¹⁸⁾ with slight modifications. In brief, cerebrum specimens were dispersed using a pipette. Aliquots (0.1 mL) of the cell suspension, usually 2×10⁵ cells/mL, were placed in 96-well plates pre-coated with 0.1% poly-DL-ornithine. After incubation for 60 min at 37°C in a 5% CO₂ atmosphere, the medium was changed to serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing N₁ constituents, consisting of 5 ng/mL of insulin, 5 ng/mL of transferrin, 7.3 ng/mL of progesterone, 16 µg/mL of putrescine, and 5 ng/mL of selenium, then the cells were successively incubated for 3 d under the same conditions described above for the neuron-enriched cultures. Contamination by non-neuronal cells in the cultured cell populations, estimated by determination with anti-β-III tubulin antibodies, was scarcely observed until day 7 after inoculation.

In separate experiments, aliquots (0.1 mL) of the cell suspension detailed above were placed in 96-well plates pre-coated with 0.1% poly-DL-ornithine. After incubation for 60 min at 37°C in a 5% CO₂ atmosphere, the medium was changed to DMEM containing 5% fetal calf serum (FCS). The next day, the medium was changed to that containing 20 µM cytosine arabinoside, and the cells were successively incubated for 3 d (mixed neuronal and glial cultures). The time of conversion to FCS was designated as day 0. The populations of neuronal cells in the culture dishes ranged from about 50–60% on day 5 after inoculation, as estimated by the ratio of cells reacting with anti-β-III tubulin antibodies to the total number of cells, with about 10% of the total cells stained with anti-glial fibrillary acidic protein (GFAP) (astrocyte-specific marker) antibodies.

Cultured cells were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS(−) for 10 min, and treated with Blocking One solution (Nakalai, Japan). Next, they were incubated for 2 h with anti-β-III tubulin (Sigma, U.S.A.; 1 : 500) or GFAP (Becton Dickinson U.S.A.; 1 : 1000) primary antibodies at 4°C, then washed in TTBS (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05% Tween20) and incubated for 2 h with fluorescent-conjugated secondary antibodies (Alexa fluoro546, Molecular Probes, U.S.A.; 1 : 1000) at room temperature. The cells were observed using an IX70 fluorescence microscope (Olympus, Japan).

MTT Reduction Assay of Primary Cultured Cells

An MTT reduction assay assesses the integrity of mitochondrial functions, as described by Mosmann.¹⁹⁾ This assay is based on the principle that the tetrazolium ring on MTT is reduced by the electron transfer system in active mitochondria. The primary enzymes to reduce MTT have been shown to be succinate dehydrogenase in complex II (succinate: ubiquinone oxidoreductase) and antimycin A-sensitive cytochrome c oxidase in complex III.²⁰⁾ In the present study, the assay was performed according to the specifications of the manufacturer (MTT kit I; Roche, Mannheim, Germany).

The contents of DNA in the cells were determined according to a method previously described.¹⁶⁾ In brief, 2×10⁶ cells were cultured in 24-well plates for 3 d, then solubilized in 1 mL of lysis buffer, and treated with a proteinase K and RNase A solution. DNA was extracted twice with phenol–chloroform and precipitated twice, first with isopropyl alcohol and then with 70% ethanol, after which air-dried pellets were solubilized. DNA concentrations were calculated by determining the OD₂₆₀ value.

Results

Comparison of Effects of Nitroprusside with Those of Other Inhibitors on Aconitase Activities

Aconitase was obtained from a commercial source in an inactive state and required activation, as a previous study indicated that Fe(II) is needed in the presence of a reducing agent such as cysteine and dithiothreitol for that activation.³⁾ In the present study, commercial porcine heart aconitase ([3Fe–4S] form) was activated for 20 min at 37°C in the presence of Fe(II)AS (0.05 mM) and dithiothreitol (2.5 mM), then enzyme activity was determined in the presence of the various inhibitors.

The effects of the various inhibitors containing Fe chelators and oxidants on aconitase activities were examined in the presence of Fe(II)AS and dithiothreitol. As shown in Fig. 1, the Fe-chelators, ethylenediamine tetraacetic acid (EDTA) and deferoxamine,²¹⁾ strongly inhibited the activity, while fluorocitrate (FC: a competitive inhibitor),²²⁾ cyanide ion (CN⁻: a noncompetitive inhibitor, a heme poison),²³⁾ ferricyanide ([Fe(III)(CN)₆]),^13,24,25) and various oxidants including superoxide anion,^2,13,26,27) APS,³⁾ and hydrogen peroxide²⁾ showed moderate inhibition. In contrast, only sodium nitroprusside (SNP) ([Fe(II)(CN)₅(NO⁺)])²⁸⁾ significantly increased the enzyme activity.

Fig. 1. Effects of Various Inhibitors on Aconitase Activity in Presence of Fe(II)AS

The effects of various inhibitors on activated aconitase were examined in the presence of Fe(II)AS (0.05 mM). Aconitase was activated at 37°C for 20 min in the presence of Fe(II)AS (0.1 mM), then inhibitory activity was determined at room temperature using a UV assay. FC, EDTA, Def and hydrogen peroxide were used at a concentration of 0.1 mM, while the concentrations of SNP [Fe(CN)₅NO], APS, ferricyanide [Fe(CN)₆], and KCN were 0.05, 0.25, 2, and 5 mM, respectively. X/XO: superoxide anion generated by pre-incubation at 37°C for 20 min with hypoxanthine (X) (0.25 mM) and xanthine oxidase (XO) (125 m unit/mL); FC: D,L-fluorocitrate, Def: deferoxamine, APC: ammonium peroxodisulfate. Blank: activity only in the presence of DTT. C: control, activity in the presence of DTT and Fe(II)AS. Data are expressed as the mean±S.E.M. (bar). n=3–5, * p<0.01 to control (one-way ANOVA followed by a Tukey–Kramer multiple comparisons test).

The aconitase-inhibiting activities of the inhibitors except for SNP were similar to those noted in previous reports,³⁾ though the inhibition rates of oxidants were low because of the protective effects of Fe(II) on aconitase. It is known that SNP also generates NO, even though this molecule has a ferricyanide moiety, as well as sodium ferricyanide, a strong inhibitor of aconitase. It was previously reported that NO did not inhibit aconitase at low concentrations, while high concentrations led to inactivation of m-aconitase.¹²⁾ Furthermore, in addition to the NO group, the SNP molecule contains Fe moiety, that is released in an ionic form on disintegration,²⁴⁾ thus there is a possibility that the increasing effects exerted by SNP on aconitase are due to Fe ions. Therefore, we examined the role of the NO group in this molecule and its effects on aconitase activity.

Effects of SNP and Other NO-Donors on Activation of Aconitase

The effects of SNP and other NO-donors, on activation of aconitase were investigated to determine whether the promoting effect of SNP is due to NO or Fe present in the molecule. Moreover, NO-donors are known to have at least 3 groups, nitrosonium ion (NO⁺), nitric oxide (NO*), and peroxynitrite (ONOO⁻), under different redox conditions, which have markedly different biologic effects.^29,30) Notably, it has been reported that the presence of ascorbic acid or another reducing agent converts SNP from NO⁺ species to NO* species.³⁰⁾ It was also shown that a NO-donor, 3-morpholino-sydnonimine (SIN), generates NO and superoxide anion,³¹⁾ apparently resulting in the formation of peroxynitrite, a potent inhibitor of aconitase.³²⁾ Therefore, in addition to SNP (rapid NO-releasing chemical), SIN, Deta NONOate (NOC18)^6,10) (a very slow NO-releasing donor), and NaNO₂ (the most popular NO-releasing compound) were chosen as NO-donors in the present study.

Figure 2A shows that all NO-donors including SNP promoted aconitase-activation in the presence of Fe(II)AS, whereas only SNP increased it slightly even in the absence of Fe(II)AS. Fe released from SNP may be responsible for the observed effects on activation of aconitase. Figure 2B indicates that incubation of aconitase with all NO-donors resulted in time-dependent promotion of aconitase-activation in the presence of Fe(II)AS. This activation was significantly promoted by the NO-donors as compared with Fe(II)AS alone. As shown in Fig. 2C, SNP strongly promoted aconitase-activation in a dose-dependent manner in the presence of Fe(II)AS, while that promotion was dose-dependent and moderate in its absence. Activation by SNP alone may be mediated by Fe ion released from the SNP molecule. Indeed, pre-incubation of SNP (0.025 mM) with dithiothreitol (DTT) (2.5 mM) at 37°C led to time-dependent activation as compared to without pre-incubation (data not shown). Figure 2D demonstrates that both SIN and NOC18 strongly promoted that activation in a dose-dependent manner in the presence of Fe(II)AS, whereas they had no effect in its absence. NaNO₂ also strongly promoted Fe-dependent activation in a dose-dependent manner, though higher concentrations (ca. 10 mM) of NaNO₂ were required for that effect (data not shown). The promotions of aconitase-activation by NO-donors were also confirmed by using a coupling method for aconitase activity (data not shown).

Fig. 2. Effects of NO-Donors, SNP, SIN, NaNO₂, and NOC18, on Aconitase-Activation

(A) Effects of SNP, SIN, NaNO₂, and NOC18 on aconitase-activation. The effects of SNP (0.1 mM), SIN (2 mM), NaNO₂ (2 mM) and NOC18 (1 mM) on aconitase-activation were determined with 2.5 mM of DTT in the presence (0.05 mM) or absence of Fe(II)AS. All of the tested NO-donors promoted aconitase-activation in the presence of Fe(II)AS. Data were obtained from 3 experiments and are expressed as the mean±S.E.M. (bar). B: Blank, activity only in the presence of DTT. C: control, activity in the presence of DTT and Fe(II)AS. * p<0.01, ** p<0.05 vs. control (one-way ANOVA followed by a Tukey–Kramer multiple comparisons test). (B) Time course of activation of aconitase by SNP (0.1 mM), SIN (0.1 mM), NaNO₂ (2 mM) and NOC18 (1 mM) in the presence of Fe(II)AS (0.05 mM) and DTT (2.5 mM). (C) Concentration-dependent activation of aconitase by SNP in the presence of Fe(II)AS. Incubation of aconitase with SNP along with 2.5 mM of DTT resulted in dose-dependent promotion of aconitase-activation in both presence (0.02 mM) and absence of Fe(II)AS. (D) Concentration-dependent activation of aconitase by SIN and NOC18 in presence of Fe(II)AS. Incubation of aconitase with SIN and NOC18 promoted dose-dependent activation only in the presence of Fe(II)AS (0.02 mM) and DTT (2.5 mM). control: activity in the presence of DTT and SIN or NOC18. Data were obtained from 3 experiments and are expressed as the mean±S.E.M. (bar).

These findings suggest that NO released from all of the tested NO-donors promotes activation of aconitase in the presence of Fe(II).

Effects of NO-Scavengers on Aconitase-Activation Promoted by NO

Since NO reacts with superoxide anion, yielding peroxynitrite, it was also of interest to explore the effects of NO under conditions generating superoxide anion. Superoxide was generated by pre-incubation at 37°C for 20 min with hypoxanthine (X) and xanthine oxidase (XO).^26,27) The effects of NO-donors on aconitase-activation were examined in the presence of X/XO, with the results shown in the Fig. 3A. X/XO strongly counteracted the promoted activation by SIN and NOC18, and moderately decreased that by NaNO₂, whereas it only slightly inhibited that by SNP. Fe ions released from the SNP molecule were estimated to become reinserted into aconitase damaged by ONOO⁻, as Fe ion can reinsert into aconitase damaged by various oxidants.³⁾ However, that mechanism is unclear.

Fig. 3. Effects of NO-Scavengers on Aconitase-Activation Promoted by NO-Donors

(A) Effects of X/XO on aconitase-activation promoted by NO-donors. Hypoxanthine (0.25 mM) was pre-incubated with xanthine oxidase (125 m unit/mL) for 20 min at 37°C, then incubated with aconitase for 10 min at 37°C immediately after adding the reaction mixtures in the presence of Fe(II)AS (0.02 mM) and SNP (0.1 mM), SIN (2 mM), NaNO₂ (2 mM), and NOC18 (0.2 mM). Aconitase activity was determined as described in Materials and Methods. Data were obtained from 3–6 experiments and are expressed as the mean±S.E.M. (bar). Fe: activity in the presence of DTT and Fe(II)AS. X/XO: activity in the presence of hypoxanthine and xanthine oxidase. * p<0.01, ** p< 0.05 vs. (Fe alone) (two-way ANOVA followed by a Tukey–Kramer multiple comparisons test). (B) Effects of heme and PTIO on aconitase-activation promoted by NO-donors. NO is known to be counteracted by heme and PTIO. The effects of heme (0.05 mM) and PTIO (1 mM) on NO-donor-mediated promotion of aconitase-activation were examined in the presence of Fe(II)AS (0.02 mM) and SNP (0.025 mM), SIN (1 mM), NaNO₂ (10 mM), and NOC18 (0.2 mM). Data were obtained from 3–4 experiments and are expressed as the mean±S.E.M. (bar). B: Blank, activity only in the presence of DTT. C: control, activity in the presence of DTT and Fe(II)AS. N: none, without TPIO and Heme. * p<0.01, ** p<0.05 vs. control or N (two-way ANOVA followed by a Tukey–Kramer multiple comparisons test).

NO is also known to be counteracted by various NO-scavengers including heme³³⁾ and imidazolineoxyl N-oxide derivative (PTIO).³⁴⁾ Therefore, the effects of heme and PTIO on NO-mediated promotion of aconitase-acitivation were examined. As shown in Fig. 3B, the promoting effect of all tested NO-donors on activation was completely prevented by PTIO, while that was moderately prevented by heme. Taken together, these findings suggest that NO released from NO-donors promotes aconitase-activation.

Fe-Dependence for Promotion of Aconitase-Activation by NO-Donors

Our findings clearly indicate that NO strongly promoted Fe-dependent activation of aconitase in time- and concentration-dependent manners. However, the relationship of NO and Fe ion during promotion of aconitase-activation is yet to be clarified. Therefore, we used various concentrations of Fe(II)AS during activation of aconitase in the presence and absence of the NO-donors, SNP, SIN, NaNO₂ and NOC18. As shown in Fig. 4A, Fe(II)AS caused a dose-dependent promotion of aconitase-activation at lower concentrations in the presence of all tested NO-donors as compared to Fe(II)AS alone. However, the maximum levels of aconitase-activation in the presence of each donor was nearly the same as with Fe(II)AS alone.

Fig. 4. Fe-Dependence for Promotion of Aconitase-Activation by NO-Donors

(A) Effects of Fe-concentration on aconitase-activation in the presence of NO-donors. Various concentrations (0–0.2 mM) of Fe(II)AS were added during activation of aconitase in the presence of SNP (0.025 mM), SIN (0.5 mM), NaNO₂ (5 mM) and NOC18 (0.1 mM), and their absence. Data were obtained from 3–6 experiments and are expressed as the mean±S.E.M. (bar). (B) Effects of Fe(II)AS (0–0.1 mM) and SNP (0–0.1 mM) on aconitase-activation in the presence of SIN (1 mM) and its absence. Incubation of aconitase with Fe(II)AS (0–0.1 mM) resulted in dose-dependent promotion of aconitase-activation in the presence of SIN (1 mM), while that with SNP (0–0.1 mM) and SIN (1 mM) had nearly the same effects as with SNP alone. Data were obtained from 3–4 experiments and are expressed as the mean±S.E.M. (bar). (C) Effects of [Fe(II)(Lactate)] (0–0.1 mM) and [Fe(III)(Citrate)] (0–0.1 mM) on aconitase-activation in the presence of SIN (1 mM) and its absence. Incubation of aconitase with [Fe(II)(Lactate)] (0–0.1 mM) and SIN (1 mM) resulted in dose-dependent promotion of aconitase-activation, while that with [Fe(III)(Citrate)] (0–0.1 mM) and SIN (1 mM) had no effect. Data were obtained from 3 experiments and are expressed as the mean±S.E.M. (bar). (D) Effects of [Fe(II)(β-CG)] complex on aconitase-activation in the presence of SIN (1 mM) and its absence. Incubation of aconitase with the [Fe(II)(β-CG)] complex resulted in significant promotion of aconitase-activation in the presence of SIN (1 mM). Data were obtained from 3 experiments and are expressed as the mean±S.E.M. (bar). * p<0.01 vs. [Fe(II)(β-CG)] complex alone (two-way ANOVA).

Next, we examined whether NO utilizes bound Fe in various Fe-complexes during aconitase-activation. It has been reported that NO predominantly reacts with Fe(III) or Fe(II) ions in heme, Fe-S centers in various enzyme proteins, and ferritin.^4,5) On the other hand, it has also been reported that there is virtually no free Fe in cells.³⁵⁾ Therefore, SNP was initially chosen for the Fe-complex. The SNP concentration varied, whereas when SIN was used as a NO-donor, the concentrations were kept constant (1 mM). As shown in Fig. 4B, SNP showed only a slight increase in aconitase-activation in both the presence and the absence of SIN, while Fe(II)AS caused a dose-dependent promotion of that activation in the presence of SIN. These findings suggest that NO can not utilize Fe in the SNP molecule for aconitase-activation.

We also examined the effects of [Fe(II)(Lactate)] and [Fe(III)(Citrate)] as Fe-complexes on aconitase-activation in the presence and the absence of SIN. As shown in Fig. 4C, incubation of aconitase with [Fe(II)(Lactate)] resulted in a dose-dependent promotion of aconitase-activation in the presence of SIN, whereas that with [Fe(III)(Citrate)] had no effects. The complex could not promote activation even when a high concentration (20 mM) of the reducing compound, DTT, was used to reduce Fe(III) in the complex (data not shown). Moreover, [Fe(II)(Citrate)] and [Fe(II)(Glutamate)] complexes prepared from long-term incubations of citrate and glutamate with Fe(II)O powder did not activate the [3Fe–4S] form of aconitase, as shown in our previous study.¹⁶⁾ In addition, [Fe(II)(Gluconate)] showed nearly same activity as [Fe(II)(Lactate)] in the presence of SIN (data not shown). These findings suggest that NO dose not utilize strongly bound Fe such as that in the [Fe(III)(Citrate)] complex.

Recently, we found that β-citrylglutamate (β-CG), isolated from newborn rat brains, was an endogenous low molecular weight Fe chelator,³⁶⁾ while the [Fe(II)(β-CG)] complex plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it.¹⁶⁾ Therefore, we used the[Fe(II)(β-CG)] complex as an Fe-complex containing moderately bound Fe in our experiments. As shown in Fig. 4D, [Fe(II)(β-CG)] promoted dose-dependently aconitase-activation in the presence of SIN (1 mM), while it activated that only moderately without SIN as previously described.¹⁶⁾

These findings suggest that NO essentially requires Fe(II) ion for binding to itself for aconitase-activation.

Questions regarding why contradictory results have been reported showing that NO either does or does not inactivates aconitase have not been answered. To address this issue, we prepared Fe-free activated aconitase. Aconitase was activated by Fe(II)AS (100 µM) at 37°C for 10 min. To remove excess low molecular activating agents such as Fe ion, aconitase protein preparations were rapidly desalted using a Bio-Gel p-6 cartridge (1.6×4 cm) equilibrated with 50 mM Tris–HCl buffer, pH 7.4, containing 0.5 mM dithiothreitol, then the effects of the NO-donors were immediately examined using the Fe-free aconitase preparations. As shown in Table 1, SIN and NOC18 inactivated aconitase at 4 mM, whereas SNP activated that at a low concentration (0.1 mM), and inactivated it at a high concentration (0.4 mM). NaNO₂ had no effect at either low or high concentrations, whereas it inactivated the enzyme at very high concentrations (>10 mM) (data not shown). Activation of aconitase by SNP at low concentrations may be due to Fe ion released from SNP molecules during incubation in the presence of DTT. The inactivation of aconitase by NO-donors seen in the present study agrees with previously reported in vitro studies.⁶⁾ Interestingly, the activities of these Fe-free aconitase were strongly promoted by adding Fe(II)AS in the presence of all NO-donors, though the degrees of promotion varied by type of donors. Our findings suggest that NO inhibits aconitase activity at high concentrations in the absence of Fe(II), whereas it has either no effect apparently or promotes Fe-dependent activation of the enzyme in the presence of Fe(II).

Table 1. Effects of NO-Donors on Activated Aconitase in Presence and Absence of Fe(II)AS

NO-donors	Aconitase activity (% of control)
	0.1 mM	0.4 mM	Added Fe
SNP	174.4±39.5*	22.5±1.0*	126.7±3.5*
	1 mM	4 mM	Added Fe
SIN	97.8±5.4	33.8±2.8*	194.0±3.5*
NaNO2	97.4±2.3	107.5±7.5	1553.0±12.8*
NOC18	60.6±4.0*	30.3±1.0*	662.8±8.0*

Commercial aconitase (purified from pig hearts) was activated by Fe(II)AS and dithiothreitol, then the enzyme preparation was rapidly desalted using a Bio-Gel p-6 cartridge. Fe-free activated aconitase was pre-incubated for 20 min at 37°C in the presence of the NO-donors and enzyme activity was determined as described in Materials and Methods. In separate experiments, activated aconitase was pre-incubated in the presence of both the NO-donors and Fe(II)AS (0.05 mM). The concentrations of the NO-donors used were at 0.1 mM of SNP, 2 mM of SIN, 2 mM of NaNO₂, and 1 mM of NOC18. Data are expressed as the mean±S.E.M. (bar). Asterisk indicates significant difference with value for control (100%) (* p<0.01, n=3, two-way ANOVA followed by a Tukey–Kramer multiple comparisons test).

Effects of NO-Donors on Aconitase-Reactivation in Mitochondria Treated with APS

Aconitase activity in mitochondria has been reported to consist of a sensitive redox sensor of reactive oxygen and nitrogen species in cells.¹⁷⁾ It has been shown that aconitase is inhibited by various oxidants such as H₂O₂ by damaging Fe_a in [4Fe–4S] aconitase clusters.²⁾ Also, APS is known to inactivate aconitase as a relatively mild oxidant as compared with H₂O₂.³⁾

We examined the effects of APS using intact mitochondria prepared from rat hearts. As shown in Fig. 5A (blank column) the aconitase activity decreased when APS was added into mitochondria. Therefore, we determined whether the present NO-donors had effects to reactivate aconitase damaged by APS. Intact mitochondria suspensions were pre-incubated with APS for 10 min at 37°C, followed by incubation with the NO-donors in the presence or absence of Fe(II)AS for 10 min at 37°C. The mitochondria were then solubilized in 0.5% Triton X-100 and aconitase activities in the solution were determined. As shown in Fig. 5A, all of the tested NO-donors promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II)AS (0.04 mM), but not in its absence (data not shown). However, the degrees of reactivation of aconitase by SIN and NaNO₂ were relatively lower than that by SNP and NOC18.

Fig. 5. Treatments of Mitochondria with Oxidants, Effects of NO-Donors on Aconitase-Reactivation

(A) Effects of NO-donors on reactivation of aconitase damage by APS. Intact mitochondria suspensions were pre-incubated with APS (0.1 mM) for 10 min at 37°C, followed by incubation with the NO-donors in the presence of Fe(II)AS (0.04 mM) or its absence for 10 min at 37°C. Mitochondria were then solubilized in 0.5% Triton X-100, and aconitase activities in the solution were determined within 30 s. SNP, SIN, NaNO₂, and NOC18 were used at 0.02–0.1 mM, 0.4–2 mM, 4–20 mM, and 0.08–0.4 mM, respectively. Blank: absence of Fe(II)AS, control: presence of Fe(II)AS. Data were obtained from 3–6 experiments and are expressed as a percentage of the control, [mean±S.E.M. (bar)]. * p<0.01 vs. control (one-way ANOVA followed by a Tukey–Kramer multiple comparisons test). (B) Effects of NO-scavengers on reactivation of aconitase promoted by NO-donors. The effects of NO are counteracted by NO-scavengers such as PTIO. The effects of PTIO (1 mM) on NO-donor-mediated promotion of aconitase-reactivation were examined in the presence of Fe(II)AS (0.02 mM) and SNP (0.025 mM), SIN (2 mM), NaNO₂ (10 mM), and NOC18 (0.4 mM). Blank: absence of Fe(II)AS, control: presence of Fe(II)AS, N: none, without PTIO in the presence of Fe(II)AS. Data were obtained from 3–6 experiments and are expressed as a percentage of the control, [mean±S.E.M. (bar)]. * p<0.01 vs. N (two-way ANOVA followed by a Tukey–Kramer multiple comparisons test).

The actions of NO are known to be counteracted by NO-scavengers such as PTIO. Therefore, the effects of PTIO on NO-donor-mediated promotion of reactivation of aconitase in mitochondria were examined. As shown in Fig. 5B, the promoting effects of all the NO-donors on reactivation were prevented by PTIO. These findings suggest that NO released from NO-donors promotes reactivation of aconitase in a mitochondrial matrix.

Effects of NO-Donors on Primary Cultures of Neurons from 1-d-Old Mouse Brain

The number of mitochondria present in a cell depends on the metabolic requirements of that cell. An adequate energy supplied by mitochondria is essential for neuronal survival,³⁷⁾ and based on their role as a cellular powerhouse, mitochondria are emerging as key participants in cell survival. Furthermore, mitochondrial oxidative phosphorylation provides the major source of ATP in cortical neurons. It is known that an assay of MTT reduction activity can assess the integrity of mitochondrial function.¹⁹⁾ The resultant reaction is mainly attributed to mitochondrial enzymes and electron carriers, and can also be used to detect cell viability.^20,38) In addition, NO has been shown to have markedly different biological effects in neural cells depending on its redox state. NO has a neurotoxic effect by reacting with superoxide anion to produce peroxynitrite (ONOO⁻). In contrast, the nitrosonium ion (NO⁺) has a neuroprotective effect via S-nitrosylation of thiol groups on the N-methyl-D-aspartate receptor.^30,39) Therefore, mitochondrial functions were examined using an MTT reduction assay with neuron-enriched cultures after exposure to 0.1–1000 µM of the NO-donors, SNP, SIN, and NOC18, for the first 3 d after seeding. As shown in Fig. 6A, all NO-donors except for SNP enhanced MTT reduction activity at low concentrations in primary cultures of neurons, whereas MTT reduction activity was decreased in a dose-dependent manner with high concentrations of all of the tested NO-donors.

Fig. 6. Effects of NO-Donors on Neurons Cultured from 1-d-Old Mouse Cerebrum Tissues

Mitochondrial functions were determined using an MTT reduction assay in primary cultures of neurons exposed to 0.1–1000 µM of the NO-donors, SNP, SIN, and NOC18, for the first 3 d after seeding. Data were obtained from 3–6 experiments and analyzed for statistical significance by a two-way ANOVA. Significant differences between groups were determined using a Tukey–Kramer multiple comparisons test, with the level of significance set at * p<0.05 vs. control (100%) (A) Concentration-dependent effects of NO-donors on MTT reduction activity in neuron-enriched cultures. SIN and NOC18 significantly enhanced MTT reduction activity at low concentrations in cultures of neurons, whereas they decreased that activity at high concentrations. Contrary to the other NO-donors, SNP decreased MTT reduction activity at both low and high concentrations. (B) Concentration-dependent effects of NO-donors on MTT reduction activity in mixed neuronal and glial cultures. All of the NO-donors except for SNP significantly enhanced MTT reduction activity at low concentrations. (C) Effects of NO-donors on DNA content in mixed neuronal and glial cultures. DNA contents from cells in wells treated with the NO-donors were determined. Those contents in wells treated with SIN and NOC18 at both 10 and 30 µM were slightly increased, whereas those in wells treated with SNP were significantly decreased. (D) Dose-dependent effects of β-CG and [Fe(II)(β-CG)] on MTT reduction activity in presence of NO-donors in mixed neuronal and glial cultures. The [Fe(II)(β-CG)] showed a higher MTT reduction activity even at 50 µM in the presence of SIN, whereas β-CG had only a lower activity at 50 µM, then increased gradually the activity until 200 µM. On the contrary, Fe(II)AS decreased the activity in the presence of SIN. (E) Effects of NO-donors on MTT reduction activity in mixed neuronal and glial cultures in presence of [Fe(II)(β-CG)] complex. SIN and NOC18 significantly enhanced MTT reduction activities in the presence of the [Fe(II)(β-CG)] complex, whereas neither NO-donor had an effect in the presence of Fe(II)AS and [(Fe(II)(Citrate)]. All NO-donors were used at 30 µM, while Fe(II)AS, [Fe(II)(Citrate)], β-CG and [Fe(II)(β-CG)] at 200 µM. (—): none, Fe: Fe(II)AS, Ci: citrate, Fe-Ci: [(Fe(II)(Citrate)], Fe-β-CG: [Fe(II)(β-CG)] * p<0.05 vs. Fe-β-CG alone, ** p<0.05 vs. SIN or NOC18 alone.

Previously, the effects of NO and related nitroso-compounds were examined using mixed neural and glial cultures, and redox-based mechanisms for neuroprotective and destructive effects of NO were reported.³⁰⁾ In the present study, the effects of SNP, SIN, and NOC18 in mixed neuronal and glial cultures were examined at concentrations ranging from 0.1–1000 µM. The results obtained were nearly the same as those with neuron-enriched cultures (data not shown). Indeed, SIN and NOC18 enhanced MTT reduction activity at low concentrations, while SNP decreased it slightly even at a low concentration. However, SNP is also known to prevent chemical hypoxia-induced cell death in C6 glioma cells, though at high concentrations,⁴⁰⁾ and also upregulates m-aconitase activity and its gene expression in prostate carcinoma cells at low concentrations.²⁹⁾ Therefore, the effects of the present NO-donors on MTT reduction activities in the mixed cultures were examined at low concentrations ranging from 0.1–10 µM, with the results shown in Fig. 6B. Both SIN and NOC18 significantly enhanced MTT reduction activity in a dose-dependent manner, whereas SNP decreased it slightly.

In separate experiments, DNA contents in cultured cells treated with NO-donors were determined, with the results presented in Fig. 6C. Those DNA contents in cells treated with SIN and NOC18 at both 10 and 30 µM were slightly increased, whereas those in cells treated with SNP were significantly decreased. However, the changes in DNA contents in SIN and NOC18-treated cells were not statistically significant. In this culture system, the number of neuronal cells declined to about 70% after 3 d of culture, which was determined based on the DNA contents in the dishes. Therefore, the increase in cell number by NO-donor treatment was considered to reflect an increased number of surviving cells.

In our earlier in vitro experiment (Fig. 4D), SIN increased the activation of aconitase by about 2–3-fold with the addition of the [Fe(II)(β-CG)] complex (about 200 µM) as compared to SIN alone. Therefore, concentration-dependent effects of β-CG and [Fe(II)(β-CG)] on MTT reduction activity were examined in presence of SIN using mixed neuronal and glial cultures. As shown in Fig. 6D, the [Fe(II)(β-CG)] showed a higher MTT reduction activity even at 50 µM in the presence of SIN, whereas β-CG had only a lower activity at 50 µM, but then gradually increased the activity until 200 µM. On the contrary, Fe(II)AS decreased MTT reduction activity in the presence of SIN. NOC18 also showed nearly the same results (data not shown). In addition, SIN and NOC18 showed fairly effective promotion of MTT reduction activities in the presence of β-CG (200 µM). β-CG is considered to form an [Fe(II)(β-CG)] complex with Fe ion (total about 2 µM) in culture medium containing bovine calf serum, as we previously demonstrated that β-CG can form a relatively strong complex with Fe ion in neutral pH.³⁶⁾ Therefore, β-CG would show effect compatible to that of the [Fe(II)(β-CG)] complex.

Next, the effects of SIN, and NOC18 were examined in the presence of Fe(II)AS, citrate, [Fe(II)(Citrate)] and [Fe(II)(β-CG)] complex using mixed neural and glial cultures. As shown in Fig. 6E, SIN and NOC18 significantly enhanced MTT reduction activities in the presence of the [Fe(II)(β-CG)] complex (200 µM), whereas neither of those NO-donors had effects in the presence of Fe(II)AS, citrate and [Fe(II)(Citrate)].

In separate experiments, cultured cells were exposed to 30 µM of NOC18 for the first 3 d after seeding in the presence of [Fe(II)(β-CG)], then surviving cells were stained on day 5 with β-tubulin-III antibody (neuro-marker). Stained neurons in NOC18-treated wells in the presence of [Fe(II)(β-CG)](Fig. 7F) were more abundant than those in wells treated with [Fe(II)(β-CG)] alone (Fig. 7E). In wells treated with NOC18 alone, stained neurons were scarce (Fig. 7B). In addition, stained neurons were nearly absent in wells treated with Fe(II)AS alone or NOC18 containing Fe(II)AS (Figs. 7C, D). SIN also showed nearly the same results as NOC18, whereas SNP led to neurodestructive result (data not shown).

Fig. 7. Photomicrographs of Neurons Stained with β-Tubulin-III Antibody: Effects of NO-Donors with [Fe(II)(β-CG)] Complex in Mixed Neuronal and Glial Cultures

Cultured cells were exposed to 30 µM of NOC18 for the first 3 d after seeding in the presence of [Fe(II)(β-CG)], then surviving cells were stained on day 5 with β-tubulin-III antibody (neuro-marker). Stained neurons in NOC18-treated wells in the presence of [Fe(II)(β-CG)] were more abundant than those in wells treated with [Fe(II)(β-CG)] alone. In wells treated with Fe(II)AS alone or NOC18 with Fe(II)AS, stained neurons were nearly non-existent. Bar=50 µm. (A) Control culture. (B) Culture with NOC18 (30 µM). (C) Culture with Fe(II)AS (200 µM). (D) Culture with NOC18 (30 µM) and Fe(II)AS (200 µM). (E) Culture with [Fe(II)(β-CG)] (200 µM). (F) Culture with NOC18 (30 µM) and [Fe(II)(β-CG)] (200 µM).

These findings suggest that NO promotes utilization of the [Fe(II)(β-CG)] complex by cells and increases the number of surviving neurons by enhancing mitochondrial functions.

Discussion

NO-mediated inactivation of m-aconitase has been reported to occur in a variety of mammalian cells,^7–11) though results from in vitro studies are somewhat contradictory. Indeed, results of experiments with porcine heart m-aconitase revealed that low concentrations of NO did not inactivate aconitase, whereas high concentrations led to moderate inhibition.¹²⁾ In the present study, we anticipated that SNP ([Fe(III)(CN)₅NO]) would inhibit the enzyme due to its ferricyanide moiety, because it is known that cyanide ion and ferricyanide ([Fe(III)(CN)₆]) have such inhibition activity. However, SNP significantly increased aconitase activity. Therefore, it is reasonable to assume that NO released from the SNP molecule promotes Fe-dependent activation of aconitase. The other tested NO-donors (SIN, NaNO₂ and NOC18) promoted activation of m-aconitase in time- and dose-dependent manners in the presence of Fe(II)AS. Also, the promoting effects of the NO-donors on aconitase-activation were prevented by NO-scavengers including heme and PTIO. These findings suggest that NO released from NO-donors promotes Fe-dependent activation of aconitase.

It is important to ask why it has been reported that NO either inhibits aconitase activity or has no effect. In the present study, we prepared Fe-free activated aconitase and examined the effects of NO-donors. As shown in Table 1, SIN and NOC18 inhibited aconitase activity, which agreed with in vitro studies previously presented.^6,12) In addition, the activity of Fe-free aconitase was promoted by addition of Fe(II)AS to each of the NO-donors. These findings indicate that NO induced inhibition or promotion of aconitase activity is depend on the existence of useful Fe(II) ion in the area.

In intact mitochondria, all of the present NO-donors also promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II)AS, whereas they did not in its absence. The promoting effects of the NO-donors on the reactivation of aconitase were also prevented by the NO-scavenger, PTIO.

In addition, our preliminary experiments revealed that cytosolic aconitase (c-aconitase), iron regulatory protein 1 (IRP1), after damage with EDTA/Fe(CN)₆ reagents⁴¹⁾ was also reactivated by simultaneous addition of the [Fe(II)(β-CG)] complex with the NO-donors. However, c-aconitase prepared from rat liver cytosol had a relatively stable [4Fe–4S] cluster form, whereas c-aconitase ([3Fe–4S] cluster form) after damage with EDTA/Fe(CN)₆ was unstable due to the process of disassembly in the cluster. Therefore, the activating conditions of c-aconitase were difficult to study in greater detail.

In mixed neuronal and glial cultures, all of the NO-donors except for SNP significantly enhanced cell viability at low concentrations by promoting MTT reduction activity, whereas high concentrations decreased MTT reduction activity. It is unclear why only SNP did not enhance MTT reduction activity in the cultured cells. The SNP molecule contains Fe and cyanide moiety in addition to the NO group. It is known that Fe ion is a potent neurotoxin,⁴²⁾ though it is also an essential nutrient. This fact may explain the ineffective action of SNP. Another explanation might be by the release of cyanide ion from SNP. Roncaroli et al.⁴³⁾ reported that the release of NO from SNP in biological media does not originate from [Fe(II)(CN)₅NO]³⁻ produced on reduction of SNP but probably proceeds through the release of cyanide and further reactions of the [Fe(II)(CN)₄NO]²⁻ ion. The toxic effects of cyanide have traditionally been attributed to inhibition of cytochrome c oxidase, the terminal enzyme of the respiratory chain, although it was also reported that high levels of exogenous NO attenuated cyanide inhibition of both cytochrome c oxidase and respiration, whereas low-level NO enhanced the cyanide inhibition.⁴⁴⁾ The ineffective action of SNP toward MTT reduction may be attributed to Fe ion and/or cyanide ion released from SNP molecule. Taken together, these findings suggest that NO is also an endogenous low molecular weight Fe chaperone for aconitase, although it can take off Fe ion from the enzyme.

It is now clear that NO can either induce necrosis or apoptosis, or even protect cells from death. However, the factors that determine these paradoxical actions are largely unknown. Many of the reported contradictory results regarding damaging vs. protective actions of NO have been due to the redox status of NO in neuronal,³⁰⁾ prostate carcinoma²⁹⁾ and erythroleukemia cells,³⁹⁾ cell type such as macrophages, neuronal cells, or hepatocytes, and the level of NO exposure.⁴⁵⁾ However, Fe content in cells is considered to be another key factor for determining the effects of NO on cell viability. Recently, Kim et al.⁴⁵⁾ suggested that the hepatic cell content of non-heme Fe determines whether cytotoxic levels of NO result in apoptosis or necrosis. Indeed, NO-donors induced apoptosis in murine macrophages with low non-heme Fe contents, whereas it did not do so in hepatocytes with high non-heme Fe contents. However, pre-treatment of macrophages with FeSO₄ increased intracellular Fe to a level similar to that in hepatocytes and delayed NO-induced cell death. Moreover, NO-mediated DNA damage is known to induce apoptotic cell death in tumor cells.¹⁰⁾ Indeed, simultaneous addition of Fe-containing compounds such as SNP and FeCN protected tumor cells from NO-mediated growth inhibition and apoptosis. Taken together, these findings suggest that elevated intercellular iron rescues macrophages and tumor cells from NO-mediated growth inhibition and apoptosis.

In the present study, SIN and NOC18 enhanced MTT reduction activities in mixed neural and glial cultures, while simultaneous addition of the [Fe(II)(β-CG)] complex significantly enhanced those activities, and neither of those NO-donors had effects in the presence of Fe(II)AS and [Fe(III)(Citrate)] (Fig. 6E). Interestingly, the number of surviving neurons in NOC18-treated wells in the presence of [Fe(II)(β-CG)] was greater than in wells with NOC18 or [Fe(II)(β-CG)] alone (Figs. 7B, E, F), whereas neurons were not found in nearly all wells treated with Fe(II)AS alone or NOC18 with Fe(II)AS (Figs. 7C, D). It was previously reported that NO generated from the NO-donor, SIN or S-nitrosocysteine, led to neurotoxicity in mixed neural and glial cultures similar to the present culture systems, though the concentration of NO-donor used in that study was relatively high.³⁰⁾ In addition, we previously showed that the [Fe(II)(β-CG)] complex itself can transfer Fe to aconitase ([3Fe–4S] cluster form) after disassembly by APS in in vitro experiments including mitochondria and in cultured cells.¹⁶⁾ Therefore, these findings suggest that simultaneous addition of the [Fe(II)(β-CG)] complex is a key to determine the neuroprotective or destructive effects of NO. Taken together, we speculate that NO promotes utilization of the [Fe(II)(β-CG)] complex in cells and increases the number of surviving neurons by increasing mitochondrial functions.

Finally, NO which has a higher affinity to Fe ion, is known to easily reach mitochondria from cytosolic or extracellular sources due to its hydrophobic nature,⁶⁾ and form dinitrosyl-iron complexes in vitro,¹⁴⁾ in macrophages⁴⁵⁾ and tumor cells.⁴⁶⁾ Therefore, since the [Fe(II)(β-CG)] complex have a higher affinity to aconitase in mitochondria and can insert its Fe into the [3Fe–4S] cluster of aconitase,^16,36) it is considered that NO reacts with the Fe moiety of the [Fe(II)(β-CG)] complex, then forms an [(NO)Fe(II)(β-CG)] complex and reaches to mitochondria, resulting in activation of aconitase. β-CG seems to function as a tag addressed to aconitase in mitochondria, though further investigation is required to fully explain this interesting possibility.

Acknowledgment

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 21791761).

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