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TOXICOLOGY

Involvement of Na⁺-Ca²⁺ Exchanger in Intracellular Ca²⁺ Increase and Neuronal Injury Induced by Polychlorinated Biphenyls in Human Neuroblastoma SH-SY5Y Cells

Department of Neuroscience, Unit of Pharmacology, School of Medicine, University "Politecnica delle Marche," Ancona, Italy (S.M., P.C., G.C., S.A.); and Unit of Pharmacology, Department of Neuroscience, School of Medicine, University of Naples "Federico II", Naples, Italy (A.S., G.D.R.)

Received May 2, 2005; accepted July 7, 2005.

	Abstract

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In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10-50 µg/ml) intracellular calcium concentration ([Ca²⁺]_i) increase. Two rather specific sodium-calcium (Na⁺-Ca²⁺) exchanger (NCX) inhibitors, bepridil (10 µM) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 µM), reduced A1254-induced [Ca²⁺]_i increase. A 24-h exposure to 30 µg/ml A1254 caused remarkable SH-SY5Y neuroblastoma cell damage. It is noteworthy that both bepridil and KB-R7943 counteracted A1254-induced neuronal injury. These results indicate that NCX contributes to [Ca²⁺]_i increase and neuronal injury induced by A1254. RT-PCR experiments revealed in SH-SY5Y neuroblastoma cells the expression of NCX1 and NCX3 isoforms. To investigate which isoform was involved in [Ca²⁺]_i increase and neuronal damage induced by A1254, we used specific antisense oligodeoxynucleotides (ODNs) to reduce NCX1 or NCX3 protein expression. The results showed that only NCX1 ODN reduced [Ca²⁺]_i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca²⁺]_i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells.

The Na⁺-Ca²⁺ exchanger (NCX) is a plasmamembrane exchanger mainly involved in the maintenance of cytosolic Ca²⁺ homeostasis (Sanchez-Armass and Blaustein, 1987). This antiporter couples the uphill extrusion of Ca²⁺ to the entrance of Na⁺ into the cell (forward mode) and down its electrochemical gradient. However, this mechanism can also operate as a Na⁺ efflux-Ca²⁺ influx pathway, depending not only on membrane potential (Snelling and Nicholls, 1985) but also on the intracellular concentrations of Na⁺ and Ca²⁺ (Amoroso et al., 1997, 2000; Pignataro et al., 2004a,b).

In light of the role played by NCX in the maintenance of [Ca²⁺]_i homeostasis, the aim of the present study was to investigate the role played by NCX in PCBs-induced [Ca²⁺]_i perturbation and neuronal injury. For this purpose, SH-SY5Y cells were exposed to Aroclor 1254 (A1254), a commercial mixture of PCBs, and Fura-2 acetoxymethyl ester-monitored [Ca²⁺]_i and MTT-detected cell injury were evaluated in the presence or in the absence of NCX inhibitors, such as bepridil (Amoroso et al., 2000; Pignataro et al., 2004b), KB-R7943 (Iwamoto and Shigekawa, 1998), and specific NCX antisense oligodeoxynucleotides (ODNs) (Pignataro et al., 2004a).

	Materials and Methods

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Cell Culture. The SH-SY5Y cell line (purchased from the American Type Culture Collection, Manassas, VA) was cultured as monolayer in polystyrene dishes (100 mm diameter) and grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (Invitrogen), 1% L-glutamine (200 mM) (Invitrogen), 1% sodium pyruvate (100 mM) (Invitrogen), 100 IU/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen). Cells were grown in a humidified incubator at 37°C in a 5% CO₂ atmosphere. The medium was changed every 2 days. Each experiment was performed with cells (passage 40-60) in multiple well flow dishes.

Cytosolic Ca²⁺ Concentration Measurements. Just before the experiment, 2 x 10⁶ SH-SY5Y cells were detached, centrifuged, and then resuspended in 1 ml of a medium whose composition was 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 5.5 mM glucose, and 10 mM HEPES, pH adjusted to 7.4 with 1 M Tris (standard buffer). The cells were then incubated with 5 µM Fura-2 acetoxymethyl ester (Calbiochem, San Diego, CA) for 1 h at 30°C. After the loading period, the medium was diluted with 2 volumes of the same balanced salt solution, incubated at 37°C, and then washed twice before the experiment was performed (Amoroso et al., 2000). [Ca²⁺]_i values were measured in a 2-ml suspension of SH-SY5Y cells at 37°C in a quartz cuvette equipped with a magnetic stirrer bar. Fura-2 fluorescence was monitored in a PerkinElmer model 55 LS spectrophotofluorimeter (PerkinElmer Life and Analytical Sciences, Boston, MA). The excitation wavelengths were at 340 and 380 nm (bandpass, 5 nm) with emission at 509 nm (bandpass, 5 nm). [Ca²⁺]_i values were determined according to the equation of Grynkiewicz et al. (1985).

Determination of Cell Viability Evaluated as Mitochondrial Activity. Cell viability evaluated as mitochondrial activity was quantified by measuring dehydrogenase activity retained in the cultured cells using the MTT assay (Sigma-Aldrich Laborchemikalien, Seelze, Germany) (Mossman, 1983; Amoroso et al., 1999). The assay is based on the ability of living cells to convert dissolved MTT into insoluble formazan. Therefore, the amount of formazan produced is proportional to the number of living cells. In brief, SH-SY5Y cells (3 x 10⁵) were incubated in 2 ml of MTT solution (0.5 mg/ml) for 1 h in a humidified 5% CO₂ incubator at 37°C. Thereafter, the medium was removed and cells were washed twice with phosphate-buffered saline. 1 ml of dimethyl sulfoxide was then added to the cells to solubilize the formazan. The absorbance was read at 540 nm in a plate reader (Victor² 1420 multilabel counter; PerkinElmer Life and Analytical Sciences). Data are expressed as the amount of formazan produced. In control cultures, the same volume of A1254 vehicle was added. The osmotic pressure, measured by Autostat Osmometer 6030 (Arkray, Kyoto, Japan) in media containing standard buffer or A1254 (30 µg/ml) or its vehicle, was identical (302 mOsm). When experiments with oligonucleotides were performed, A1254 was added 5 h after the transfection.

RT-PCR Analysis of mRNA Expression of NCX Isoforms in SH-SY5Y Cells. Total RNA was extracted from SH-SY5Y cells by using TRIzol reagent (Invitrogen). To avoid contamination with genomic DNA, the extracted RNA was treated with 10 U/µl of RNAase-free DNAase I (Stratagene, Heidelberg, Germany) for 1 h at 37°C. The purity and integrity of RNA were checked by denaturing agarose gel electrophoresis. Two micrograms of total RNA were reverse-transcribed in the presence of oligo(dt) by using SuperScript III reverse transcriptase (Invitrogen) according to the protocol by the manufacturer. The retrotranscribed cDNAs (2 µl) were then amplified in a MJ Research PTC 2000 Peltier Thermal Cycler (MJ Research, Watertown, MA) by using the primers shown in Table 1, described previously by Quednau et al. (1997) and Papa et al. (2003). Each 50-µl reaction containing 1.25 U of TaqDNA polymerase (Eppendorf-5 Prime, Inc., Boulder, CO) and 10 pmol of each primer was amplified (30 cycles) by the following procedure: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The amplification products were visualized on 2% agarose gel electrophoresis, loading approximately half (25 µl) of each reaction volume per lane.

Oligonucleotide Transfection. One day after plating, SH-SY5Y cells were transfected with the appropriate phosphorothioate ODNs (Table 1) described previously by Pignataro et al. (2004a) using Lipofectamine 2000 (Invitrogen) according to the protocol by the manufacturer. In brief, oligonucleotides (5 µM) were dissolved in Lipofectamine (DNA/Lipofectamine 2000, 1:1) and then incubated at room temperature for 30 min before being added to the culture medium. Control dishes received Lipofectamine only. To evaluate cell transfection efficiency, ODNs were mixed with a plasmid encoding the enhanced green fluorescent protein as marker (Castaldo et al., 2004). Efficiency transfection was 90% (data not shown).

Drugs and Chemicals. All of the chemicals were of analytical grade and were purchased from Sigma. A1254 solution in isooctane (lot number M-841B, Analyte lot number NT01022; stock solution, 1000 ± 5 µg/ml) was purchased from Ultra Scientific (North Kingstown, RI), bepridil was obtained from Sigma, and KB-R7943 was obtained from Tocris Cookson Inc. (Bristol, UK). Bepridil was dissolved in a mixture of acetone and water (stock solution, 10 mM), whereas KB-R7943 was dissolved in a mixture of dimethylsulfoxide and water (stock solution, 10 mM).

Statistics. Data were analyzed by one-way ANOVA followed by Dunnett's test.

	Results

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Effect of Different Concentrations of A1254 on [Ca²⁺]_i in the Presence and Absence of Extracellular Ca²⁺ in SH-SY5Y Cells. When SH-SY5Y cells were exposed to different concentrations (10, 30, and 50 µg/ml) of A1254, a dose-dependent increase of [Ca²⁺]_i occurred. This increase started almost immediately and lasted for the whole considered time. The elevation of [Ca²⁺]_i induced by A1254 (30 µg/ml) was completely abolished by the removal of extracellular Ca²⁺ (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Effect of different concentrations of A1254 on [Ca2+]i in the presence and in the absence of extracellular Ca2+ ions in SH-SY5Y cells. SH-SY5Y cells were exposed to different concentrations (10, 30, and 50 µg/ml) of A1254 for 31 min. Each point represents the mean ± S.E. (bars) of four values. In the experiments performed in the absence of extracellular Ca2+ ions, A1254 was added at the concentration of 30 µg/ml. The arrow indicates the minute at which A1254 was added. Inset shows a typical calibration trace in the presence of A1254 (30 µg/ml). [Ca2+]i values were measured in sample containing 2 x 106 cells/2 ml.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of bepridil and the isothiourea derivative KB-R7943 on the [Ca2+]i elevation induced by A1254. SH-SY5Y cells were exposed to a standard medium or to A1254 in the presence or in the absence of 10 µM bepridil or 10 µM KB-R7943. Each point represents the mean ± S.E. (bars) of 4 values. The arrows indicate the minute at which NCX inhibitors and A1254 were added. [Ca2+]i values were measured in a sample containing 2 x 106 cells/2 ml.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effect of bepridil and KB-R7943 on A1254-induced neuronal injury. SH-SY5Y cells were exposed to 30 µg/ml A1254 for 24 h in the presence or in the absence of 10 µM bepridil or 10 µM KB-R7943 and then assessed in their ability to produce formazan. Each column represents the mean ± S.E. (bars) of eight values. F = 14.35 (one-way ANOVA). * denotes statistical significance (p < 0.001) versus all other experimental groups; ** denotes statistical significance (p < 0.05) versus control group.

RT-PCR Analysis of SH-SY5Y mRNA Expression of NCX1, NCX2, and NCX3. Regarding the NCX family, three dominant genes coding for three different NCX1 (Nicoll et al., 1990), NCX2 (Li et al., 1994), and NCX3 (Nicoll et al., 1996) proteins have been identified in several mammal tissues, including the central nervous system. To characterize the pattern of the expression of the three NCX isoforms in SH-SY5Y cells, RT-PCR analysis of NCX1, NCX2, and NCX3 mRNA expression was performed. As shown in Fig. 4, only mRNA expression of NCX1 and NCX3 was detected in SH-SY5Y cell line (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4. RT-PCR analysis of SH-SY5Y mRNA expression of NCX1, NCX2, and NCX3 isoforms. To characterize the pattern of the expression of the three NCX isoforms in SH-SY5Y cells, RT-PCR analysis of NCX1, NCX2, and NCX3 mRNA expression was performed. Each lane shows PCR product obtained by using NCX1, NCX2, and NCX3 isoform primers.

View larger version (31K): [in this window] [in a new window]   Fig. 5. RT-PCR analysis of NCX1 and NCX3 mRNA expression in SH-SY5Y cells transfected with respective sense and antisense ODNs. Top part of the figure shows RT-PCR of NCX1 and NCX3 products in SH-SY5Y transfected with the respective sense and antisense ODNs. Normalization of results was ensured by running parallel RT-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. Bottom part of the figure indicates optical density values representing the mean ± S.E. of three independent experiments. AS1, NCX1 antisense; AS3, NCX3 antisense.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effect of antisense NCX1 or NCX3 ODNs transfection on [Ca2+]i increase and neuronal injury elicited by A1254. A, SH-SY5Y naive and antisense NCX1 or NCX3 ODN-transfected cells were exposed to 30 µg/ml A1254. Each point represents the mean ± S.E. (bars) of four values. The arrow indicates the minute at which A1254 was added. [Ca2+]i values were measured in sample containing 2 x 106 cell/2 ml. B, SH-SY5Y naive and antisense NCX1 or NCX3 ODN-transfected cells were exposed to 30 µg/ml A1254 and assessed in their ability to reduce MTT. Each column represents mean ± S.E. (bars) of eight values. F = 14.38 (one-way ANOVA). *, p < 0.001 versus control and AS1 + A1254 groups; **, p < 0.05 versus control group; p < 0.001 versus A1254 group. AS1, NCX1 antisense; AS3, NCX3 antisense.

	Discussion

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The results of present study showed that, in SH-SY5Y neuroblastoma cells, the PCB mixture A1254 induces a dose-dependent increase of [Ca²⁺]_i that is dependent upon extracellular Ca²⁺ ions. In fact, the removal of extracellular Ca²⁺ completely abolished A1254-induced [Ca²⁺]_i increase. These results are in agreement with previous studies performed in cultured cerebellar granule cells by Mundy et al. (1999). Regarding the mechanism by which A1254 induced an influx of Ca²⁺, it has been suggested that extracellular Ca²⁺ entry occurs via L-type voltage-sensitive calcium channels (VSCC), because A1254-evoked intracellular Ca²⁺ oscillations was abolished by the addition of nifedipine (Inglefield and Shafer, 2000). We now report that NCX exchanger may be involved in Ca²⁺ entry induced by the PBC mixture. In fact, both bepridil, a rather specific inhibitor of NCX (Amoroso et al., 2000; Annunziato et al., 2004; Pignataro et al., 2004b), and KB-R7943, which inhibits NCX only when it is operating in the reverse mode (Iwamoto and Shigekawa, 1998), significantly counteracted [Ca²⁺]_i increase induced by A1254. However, bepridil has also been reported to block Ca²⁺ (Kaczorowski et al., 1989) and Na⁺ channels (Kaczorowski et al., 1989), and KB-R7943, besides its peculiar NCX-blocking properties, also exerts an inhibitory effect on several other ionic transport mechanisms, such as L-type VSCC and receptor-operated ion channels such as NMDA (Matsuda et al., 2001; Annunziato et al., 2004). Therefore, the possibility exists that these drugs' inhibition of the [Ca²⁺]_i increase induced by A1254 might be due to the blockade of Na⁺ and/or Ca²⁺ channels and/or NMDA receptor activation. On the other hand, this possibility seems to be supported by the results of Inglefield and Shafer (2000) showing that A1254-induced Ca²⁺ oscillations in developing neocortical cells can be blocked by the VSCC antagonist nifedipine, by the voltage-sensitive Na⁺ channel antagonist tetrodotoxin (Almers and Levinson, 1975; Weiser and Wilson, 2002), and by ionotropic glutamate receptor antagonists. To clarify whether NCX is effectively involved in A1254-induced [Ca²⁺]_i increase, the pattern of the expression of the three known NCX isoforms NCX1, NCX2 and NCX3 (Annunziato et al., 2004) was first characterized in SH-SY5Y cells by RT-PCR analysis. Then, the effect of A1254 on [Ca²⁺]_i increase was investigated in SH-SY5Y transfected with the antisense NCX1 or NCX3 ODNs, because only these two isoforms were detected. The results of these experiments showed that a significant reduction (60%) of [Ca²⁺]_i elevation induced by the PCB mixture occurred in cells transfected with antisense NCX1 ODN. These findings demonstrate that the NCX is effectively involved in [Ca²⁺]_i elevation induced by PCB mixture, as suggested by results obtained with bepridil and KB-R7943. In addition, it is noteworthy that, between the two NCX isoforms detected in SH-SY5Y cells, only NCX1 seems to be involved in this phenomenon. In fact, no inhibition of A1254-induced [Ca²⁺]_i increase was found in cells transfected with antisense NCX3 ODN. If the inhibition of NCX reduces A1254-induced Ca²⁺ influx, this implies that NCX is operating in the reverse mode (Ca²⁺ entry-Na⁺ efflux pathway). This mode of operation could be explained by the fact that A1254 induces a membrane depolarization, as suggested by Inglefield and Shafer (2000), with a consequent opening of Na⁺-sensitive voltage channels. This event leads to an increase of intracellular Na⁺ ion concentration, which in turn may force the NCX to work in the reverse mode (Baker and McNaughton, 1976; DiPolo, 1979; Sanchez-Armass and Blaustein, 1987; Annunziato et al., 2004). This chain of events seems to be supported by the results showing that tetrodotoxin, a well known blocker of fast-activated Na⁺ channels (Almers and Levinson, 1975; Weiser and Wilson, 2002), prevents Ca²⁺ oscillations evoked by A1254 in developing cortical neurons (Inglefield and Shafer, 2000). Thus, the hypothesis that, in SH-SY5Y cells, A1254 induces a membrane depolarization with a consequently intracellular increase of Na⁺ ions, which in turn activates NCX to operate in the reverse mode, seems reasonable.

However, the inhibition of NCX did not fully counteract [Ca²⁺]_i elevation induced by the PCB mixture. These results suggest that other mechanisms may be also involved in A1254-elicited [Ca²⁺]_i increase, such as extracellular Ca²⁺ influx through L-type VSCC (Inglefield and Shafer, 2000), inhibition of Ca²⁺-ATPase activity, Ca²⁺ sequestration by mitochondria and microsomes (Kodavanti et al., 1993), and mobilization of intracellular Ca²⁺ stores (Inglefield et al., 2001).

Another finding of the present study that deserves to be discussed is that the inhibition of A1254-elicited [Ca²⁺]_i increase mediated by NCX is able to protect SH-SY5Y cells from the injury induced by the exposure to the PCB mixture. In fact, both bepridil and KB-R7943, at the same concentrations that inhibited [Ca²⁺]_i increase, caused a significant reduction of cell injury elicited by A1254 and, in cells transfected with antisense NCX1 ODN, the extent of cell damage induced by the PCB mixture was significantly lower than that observed in naive cells. On the other hand, it is well known that an increase of [Ca²⁺]_i can trigger intracellular pathways leading to cell death (Kristian and Siesjo, 1998; Kang et al., 2002, 2004). It is noteworthy that cell protection from A1254-elicited injury obtained by the inhibition of NCX was not complete, exactly as the reduction of A1254-elicited [Ca²⁺]_i increase. In conclusion, the results of the present study seem to suggest that NCX may participate in [Ca²⁺]_i increase induced by A1254 exposure and that its inhibition may contribute to protecting cells from the injury evoked by the PCB mixture.

	Acknowledgements

 
We thank Gerardo Galeazzi, Franco Pettinari, and Carlo Alfredo Violet for their invaluable technical assistance.

	Footnotes

 
This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 2003) (to S.A. and G.D.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.088948.

ABBREVIATIONS: PCB, polychlorinated biphenyl(s); A1254, Aroclor 1254; NCX, Na⁺-Ca²⁺ exchanger; KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulfonate; ANOVA, analysis of variance; RT-PCR, reverse transcription-polymerase chain reaction; [Ca²⁺]_i, intracellular calcium concentration; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide; NMDA, N-methyl-D-aspartate; ODN, oligodeoxynucleotide; VSCC, voltage-sensitive Ca²⁺ channel(s).

Address correspondence to: Prof. Salvatore Amoroso, Department of Neuroscience, Unit of Pharmacology, School of Medicine, University "Politecnica delle Marche", Via Tronto 10/A, 60020 Torrette, Ancona, Italy. E-mail: s.amoroso{at}univpm.it

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