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

Magi, Simona; Castaldo, Pasqualina; Carrieri, Giuseppina; Scorziello, Antonella; Renzo, Gianfranco Di; Amoroso, Salvatore

doi:10.1124/jpet.105.088948

Cited by 23 publications

(24 citation statements)

References 38 publications

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“…As shown in Fig. 5A, the protein expression of NCX1 and NCX3 was detected in SH-SY5Y cells, which is consistent with their mRNA levels reported previously (Magi et al, 2005). On the other hand, only NCX1 protein expression was observed in LLC-PK 1 cells.…”

Section: Inhibitory Properties Of Ym-244769supporting

confidence: 80%

“…Antisense and sense phosphorothioate oligodeoxynucleotides, which are highly specific for NCX1 or NCX3, were synthesized as reported previously Magi et al, 2005). The sequence for each isoform was as follows: NCX1: antisense, 5Ј-TGAGACTTC-CAATTGTT-3Ј; sense, 5Ј-AACAATTGGAAGTCTCA-3Ј; NCX3: antisense 5Ј-GCCATACACAAGAG-3Ј; sense, 5Ј-CTCTTGTGTATGGC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

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YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

Iwamoto¹,

Kita²

2006

Mol Pharmacol

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We investigated the pharmacological properties and interaction domains of N- (3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide (YM-244769), a novel potent Na ϩ /Ca 2ϩ exchange (NCX) inhibitor, using various NCX-transfectants and neuronal and renal cell lines. YM-244769 preferentially inhibited intracellular Na ϩ -dependent 45 Ca 2ϩ uptake via NCX3 (IC 50 ϭ 18 nM); the inhibition was 3.8-to 5.3-fold greater than for the uptake via NCX1 or NCX2, but it did not significantly affect extracellular Na ϩ -dependent 45 Ca 2ϩ efflux via NCX isoforms. We searched for interaction domains with YM-244769 by NCX1/NCX3-chimeric analysis and determined that the ␣-2 region in NCX1 is mostly responsible for the differential drug response between NCX1 and NCX3. Further cysteine scanning mutagenesis in the ␣-2 region identified that the mutation at Gly833 markedly reduced sensitivity to YM-244769. Mutant exchangers that display either undetectable or accelerated Na ϩ -dependent inactivation, had markedly reduced sensitivity or hypersensitivity to YM-244769, respectively. YM-244769, like 2-[2-[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), protected against hypoxia/reoxygenation-induced cell damage in neuronal SH-SY5Y cells, which express NCX1 and NCX3, more efficiently than that in renal LLC-PK 1 cells, which exclusively express NCX1, whereas 2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester (SN-6) suppressed renal cell damage to a greater degree than neuronal cell damage. These protective potencies consistently correlated well with their inhibitory efficacies for the Ca 2ϩ uptake via NCX isoforms existing in the corresponding cell lines. Antisense knockdown of NCX1 and NCX3 in SH-SY5Y cells confirmed that NCX3 contributes to the neuronal cell damage more than NCX1. Thus, YM-244769 is not only experimentally useful as a NCX inhibitor that preferentially inhibits NCX3, but also has therapeutic potential as a new neuroprotective drug.

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Section: Inhibitory Properties Of Ym-244769supporting

confidence: 80%

Section: Methodsmentioning

confidence: 99%

YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

Iwamoto¹,

Kita²

2006

Mol Pharmacol

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“…Although the molecular mechanisms underlying PCB-induced neurotoxicity are still poorly understood, it has been suggested that alteration of intracellular Ca 2+ homeostasis may be involved in the PCB toxic effect (Kodavanti et al, 1993;Tilson and Kodavanti, 1998;Inglefield and Shafer, 2000;Inglefield at al., 2001). In line with this hypothesis, we previously demonstrated that the inhibition of PCBinduced elevation of intracellular Ca 2+ ([Ca 2+ ] i ) results in a reduction of neuroblastoma cell death evoked by prolonged exposure to PCBs (Magi et al, 2005).…”

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confidence: 74%

Involvement of the nitric oxide/protein kinase G pathway in polychlorinated biphenyl‐induced cell death in SH‐SY 5Y neuroblastoma cells

Canzoniero

Adornetto

Secondo

et al. 2006

J of Neuroscience Research

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Polychlorinated biphenyls (PCB) are persistent environmental contaminants whose chronic exposure can affect nervous system development and function. The cellular and molecular mechanisms underlying neuronal damage are not yet clear. In the present study, we investigated whether nitric oxide (NO) could be involved in aroclor 1254 (A1254; a PCB mixture)-induced cytotoxicity in SH-SY5Y human neuroblastoma cells. Prolonged exposure (24 hr) to A1254 (10-100 microg/ml) caused a dose-dependent reduction of cell viability that was attenuated in the presence of a calcium entry blocker, gadolinum (Gd(3+)) at 10 microM, a concentration able to block voltage-sensitive calcium channels. In addition, A1254 caused an increase of cytosolic calcium that was dependent on extracellular calcium, as measured by fura-2 videomicroscopy. A1254-induced calcium rise may stimulate NO production through an activation of neuronal NOS (nNOS). Indeed, the concomitant addition of the selective nNOS inhibitor N(omega)-propyl-L-arginine (NPLA) and A1254 prevented cell injury, suggesting that NO production plays a major role in A1254-evoked cell injury. Furthermore, the exposure (14 hr) to A1254 (30 microg/ml) produced an up-regulation of the expression of beta isoform of nNOS. This up-regulation was calcium dependent and was accompanied by an enhancement of NO production as demonstrated by an increase of nitrite formation. Moreover, A1254-induced cell injury was prevented when KT 5823, a selective cGMP/PKG inhibitor, was added concomitantly to 30 microg/ml A1254. These results suggest that PCB-induced cell death in neuroblastoma cells is mediated by an activation of the cGMP/PKG pathway triggered by NO production.

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“…Figure 1 shows the PCR amplification signals for NCX1, NCX2, and NCX3 and for the constitutive gene of GAPDH in two representative experiments of four preparations of HLM and monocytes. PCR fragments of the expected size (NCX1: 98 bp, NCX2: 137 bp, NCX3: 139 bp) were detected in the human neuroblastoma cell line SH-SY5Y used as a positive control [29]. cDNAs encoding for NCX1 and NCX3 were amplified in macrophages and monocytes at subsaturating cycle number (35 cycles).…”

Section: Resultsmentioning

confidence: 99%

“…BHK stably transfected with NCX1 (BHK-NCX1), NCX2 (BHK-NCX2), or NCX3 (BHK-NCX3) were grown in the same culture medium supplemented with 4% methotrexate [32]. SH-SY5Y cells were grown (371C, 5% CO 2 ) on plastic dishes in RPMI-1640 medium containing 10% FCS, 1% L-glutamine and 1% antibiotic-antimycotic solution [29]. …”

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Expression and function of Na⁺/Ca²⁺ exchangers 1 and 3 in human macrophages and monocytes

et al. 2009

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The Na 1 /Ca 21 exchanger (NCX) is a membrane transporter that can switch Na 1 and Ca 21 in either direction to maintain the homeostasis of intracellular Ca 21 . Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na 1 -free medium induced a significant increase in intracellular calcium concentration ([Ca 21 ] i ) in both cell types. This response was completely abolished by the NCX inhibitor 5-(N-4-chlorobenzyl)-20,40-dimethylbenzamil (CB-DMB). Moreover, inhibition of NCX activity during Ca 21 -signaling induced by histamine caused a delay in restoring baseline [Ca 21 ] i . Na 1 -free medium induced TNF-a expression and release in HLM comparable to that caused by LPS. TNF-a release induced by Na 1 -free medium was blocked by CB-DMB and greatly reduced by RNAi-mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca 21 ] i , to generate Ca 21 -signals, and to induce TNF-a production. Therefore, NCX may contribute to regulate Ca 21 homeostasis and proinflammatory functions in human macrophages and monocytes.Key words: Ca 21 -signaling . Macrophages . Monocytes . Na 1 /Ca 21 exchanger IntroductionThe Na 1 /Ca 21 exchanger (NCX) is a plasma membrane protein involved in the homeostasis of intracellular Ca 21 [1]. NCX is a bidirectional ion transporter that catalyzes the exchange of Na 1 and Ca 21 depending on their electrochemical gradients [2]. Under physiological conditions, NCX's primary role is to extrude Ca 21 from cells using the Na 1 gradient across the cell membrane (forward mode of operation) [3]. However, in some cases it can contribute to the Ca 21 influx into the cells by working in the reverse mode (i.e. coupling Ca 21 influx with Na 1 efflux) [4][5][6].Three isoforms of NCX, NCX1, NCX2, and NCX3 have been cloned in mammals, outlining a multigene family whose members share a similar molecular structure [7]. NCX is modeled with nine transmembrane segments involved in ion transport and a long central cytoplasmic loop that has regulatory functions [8]. However, truncated forms lacking certain transmembrane segments, but still possessing NCX activity, have also been Eur. J. Immunol. 2009. 39: 1405-1418 DOI 10.1002 Leukocyte signaling 1405 described as the result of alternative splicing of the three genes [9][10][11]. NCX1 is largely predominant in mammalian tissues such as heart, brain, and kidney while the expression of NCX2 and NCX3 appears to be limited to the brain and skeletal muscle [7]. NCX is a key element regulating intracellular Ca 21 concentrations ([Ca 21 ] i ) in many excitable cells, including myocardiocytes, retinal rod photoreceptors, smooth muscle cells, and neurons [3]. Non-excitable cells such as renal epithelial ce...

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

Abstract: In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10 -50 g/ml) intracellular calcium concentration ([Ca 2ϩ ] i ) increase. Two rather specific sodium-calcium (Na

Cited by 23 publications

References 38 publications

YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

Involvement of the nitric oxide/protein kinase G pathway in polychlorinated biphenyl‐induced cell death in SH‐SY 5Y neuroblastoma cells

Expression and function of Na⁺/Ca²⁺ exchangers 1 and 3 in human macrophages and monocytes

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

Abstract: In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10 -50 g/ml) intracellular calcium concentration ([Ca 2ϩ ] i ) increase. Two rather specific sodium-calcium (Na

Cited by 23 publications

References 38 publications

YM-244769, a Novel Na+/Ca2+Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

YM-244769, a Novel Na+/Ca2+Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

Involvement of the nitric oxide/protein kinase G pathway in polychlorinated biphenyl‐induced cell death in SH‐SY 5Y neuroblastoma cells

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

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

YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

Expression and function of Na⁺/Ca²⁺ exchangers 1 and 3 in human macrophages and monocytes