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

In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10–50 g/ml) intracellular calcium concentration ([Ca ]i) increase. Two rather specific sodium-calcium (Na -Ca ) exchanger (NCX) inhibitors, bepridil (10 M) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 M), reduced A1254induced [Ca ]i increase. A 24-h exposure to 30 g/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 [Ca ]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 [Ca ]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 [Ca ]i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca ]i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells. Polychlorinated biphenyls (PCB) are a class of persistent pollutants present in the environment, and there is increasing evidence that exposure to PCBs can cause neurotoxicity (Hwang et al., 2001; Mariussen et al., 2002; Howard et al., 2003; Sanchez-Alonso et al., 2003, 2004; Kang et al., 2004; Lee and Opanashuk, 2004). A number of potential mechanisms have been proposed to explain PCBs neurotoxicity [i.e., alterations in neurotransmitter levels (Bemis and Seegal, 1999), perturbation in intracellular second messenger systems (Kodavanti and Tilson, 1997; Yang and Kodavanti, 2001), and elevation of intracellular Ca concentrations ([Ca ]i) (Sharma et al., 2000). This last effect has been attributed to a Ca influx (Mundy et al., 1999; Inglefield and Shafer, 2000) or to the inhibition of Ca -ATPase activity at the synaptic level and Ca sequestration by mitochondria and microsomes (Kodavanti et al., 1993). In addition, it has been reported that PCBs can also perturb Ca homeostasis by acting on ryanodine (Wong and Pessah, 1996; Mundy et al., 1999) and inositol 1,4,5-triphosphate receptorsensitive Ca release (Inglefield et al., 2001). The Na -Ca exchanger (NCX) is a plasmamembrane exchanger mainly involved in the maintenance of cytosolic Ca homeostasis (Sanchez-Armass and Blaustein, 1987). This antiporter couples the uphill extrusion of Ca 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-Ca influx pathway, depending not only on membrane potential (Snelling and Nicholls, 1985) but also on the intracellular concentrations of Na and Ca (Amoroso et al., 1997, 2000; Pignataro et al., 2004a,b). In light of the role played by NCX in the maintenance of [Ca ]i homeostasis, the aim of the present study was to investigate the role played by NCX in PCBs-induced [Ca ]i 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 -Ca exchanger; KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulfonate; ANOVA, analysis of variance; RT-PCR, reverse transcription-polymerase chain reaction; [Ca ]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 Ca channel(s). 0022-3565/05/3151-291–296$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 88948/3052788 JPET 315:291–296, 2005 Printed in U.S.A. 291 at A PE T Jornals on July 3, 2017 jpet.asjournals.org D ow nladed from perturbation and neuronal injury. For this purpose, SHSY5Y cells were exposed to Aroclor 1254 (A1254), a commercial mixture of PCBs, and Fura-2 acetoxymethyl ester-monitored [Ca ]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), KBR7943 (Iwamoto and Shigekawa, 1998), and specific NCX antisense oligodeoxynucleotides (ODNs) (Pignataro et al., 2004a). Materials and Methods 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 g/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 Ca Concentration Measurements. Just before the experiment, 2 10 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 M 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). [Ca ]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). [Ca ]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 10) 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 phosphatebuffered 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 (Victor 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 g/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/ l 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 l) 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 50l 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 l) 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 M) 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 encod-