Role of the sodium hydrogen exchanger in maitotoxin-induced cell death in cultured rat cortical neurons

Toxicon 54 (2009) 95–102

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Role of the sodium hydrogen exchanger in maitotoxin-induced cell death in cultured rat cortical neurons Yushan Wang a, *, M. Tracy Weiss a, Junfei Yin b, Robert Frew a, Catherine Tenn a, Peggy P. Nelson a, Cory Vair a, Thomas W. Sawyer a a b

Defence Research & Development Canada-Suffield, Medicine Hat, Alberta T1A 8K6, Canada Canada West Biosciences Inc., 5429-60 Street, Camrose, Alberta T4V 4G9, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 November 2008 Received in revised form 4 March 2009 Accepted 12 March 2009 Available online 26 March 2009

Maitotoxin (MTX) is one of the most potent toxins known to date. It causes massive calcium (Ca2þ) influx and necrotic cell death in various tissues. However, the exact mechanism(s) underlying its cellular toxicity is not fully understood. In the present study, the role of the sodium hydrogen exchanger (NHE) in MTX-induced increases in intracellular Ca2þ and subsequent cell death were investigated in cultured rat cortical neurons. Intracellular Ca2þ concentrations ([Ca2þ]i) were measured fluorimetrically using FURA-2 as the fluorescence indicator. Cell death was measured with the alamarBlueÔ cell viability assay and the vital dye ethidium bromide (EB) uptake assay. Results showed that MTX increased, in a concentration dependent manner, both [Ca2þ]i and cell death in cortical neurons. Decreasing the pH of the treatment medium from 7.5 to 6.0 diminished MTXinduced cell death. The protection offered by lowering extracellular pH was not due to MTX degradation, because it was still effective even if the cells were treated with MTX in normal pH and then switched to a lower pH. Pretreatment of cells with the specific NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), prevented MTX-induced increases in [Ca2þ]i, as well as cell death in a concentration dependent manner. Furthermore, knockdown of NHE1 by SiRNA transfection suppressed MTX-induced cell death in human embryonic kidney (HEK) cells. Together, these results suggest that NHE1 plays a major role in MTX-induced neurotoxicity. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.

Keywords: Maitotoxin Ca2þ influx Neuronal cell death Sodium hydrogen exchanger Extracellular acidosis

1. Introduction Maitotoxin (MTX) is a potent marine toxin produced by the dinoflagellate Gambierdiscus Toxicus, and is a principle toxin of ciguatera seafood poisoning (Yasumoto, 2001). It evokes massive calcium (Ca2þ) increases in the intracellular space in both excitable and non-excitable cells (Cataldi et al., 1999; Escobar et al., 1998). The increased cytosolic Ca2þ triggers a variety of responses, including phosphoinositide breakdown (Berta et al., 1988; Gusovsky et al., 1989), * Corresponding author. Defence Research & Development CanadaSuffield, P.O. Box 4000, Station Main, Medicine Hat, Alberta T1A 8K6, Canada. E-mail address: [email protected] (Y. Wang).

arachidonic acid release (Choi et al., 1990), muscle contraction (Kobayashi et al., 1985; Ohizumi and Yasumoto, 1983), and neurotransmitter release (Kakizaki et al., 2006; Taglialatela et al., 1990). Maitotoxin exposure ultimately leads to necrosis in a variety of cell types, including bovine aortic endothelial cells (Estacion and Schilling, 2001); Chinese hamster ovary cells (Cataldi et al., 1999), human embryonic kidney (HEK) cells (Schilling et al., 1999b), murine macrophages (Verhoef et al., 2004), rat brain stem cells (Kakizaki et al., 2006) and rat hippocampal neurons (Zhao et al., 1999). Because of its ability to increase intracellular Ca2þ concentrations, MTX has been used as a unique pharmacological tool for research into Ca2þ dependent mechanisms (Gusovsky and Daly, 1990; Takahashi et al., 1982). However,

0041-0101/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.03.018

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the specific mechanisms by which MTX triggers Ca2þ entry and subsequent cytotoxicity have not been fully elucidated (de la Rosa et al., 2001; Sorrentino et al., 1997; Yi et al., 2006). Previous reports have shown that the actions of MTX involve the formation of a non-selective cation channel (NSCC) that resembles the purinergic P2X7 receptor and allows massive influx of Ca2þ (Lundy et al., 2004; Cataldi et al., 1999). More recently, a MTX mechanism of cell death has been proposed, that includes: (1) activation of a NSCC, permeable mainly to Naþ and Kþ, but also to Ca2þ and other divalent cations. The activation of the NSCC causes a large increase in [Ca2þ]i; (2) opening of a cytolytic/oncolytic pore that permits molecules less than 800 Da (e.g., ethidium bromide) to enter the cell; and (3) formation of a glycinesensitive cytolytic pore resulting in cell lysis and release of lactate dehydrogenase (Estacion et al., 2003; Wisnoskey et al., 2004). However, the nature of the NSCC and the exact mechanisms by which it is activated remains to be understood. Several studies have suggested the existence of a MTX receptor, which could act as an NSCC or serve as the first step in activating the NSCC (de la Rosa et al., 2007; Lundy et al., 2004; Nicolaou and Frederick, 2007). Sodium hydrogen exchangers (NHE) are a group of membrane bound proteins that are important in regulating intracellular pH, cell volume, cell growth and differentiation (Putney et al., 2002). To date, nine NHE isoforms (NHE1–9) have been identified in mammalian tissues (Luo and Sun, 2007). Among the nine NHE isoforms, NHE1 is ubiquitously expressed in virtually all mammalian cell types (Luo and Sun, 2007). In the central nervous system, NHE1 activation has been reported to play an important role in ischemic injuries, stroke and other excitotoxic events (Luo and Sun, 2007; Taylor et al., 2006). NHE inhibition by either pharmacological inhibitors or extracellular acidosis has been shown to block glutamate or hypoxia induced intracellular Ca2þ increase and subsequent cell death in cultured rat cortical neurons (Matsumoto et al., 2004; Vornov et al., 1996). In a recent report, we showed that the activation of a cell membrane sodium–calcium exchanger (NCX) in reverse mode plays an important role in MTX-evoked cell death (Frew et al., 2008). Because both NCX and NHE are involved in regulating intracellular ionic homeostasis and their functions are coupled tightly in most cell types, it is reasonable to speculate that the NHE activation may serve as a source of intracellular Naþ for NCX reversal during MTX receptor activation. The present study was undertaken to examine the role NHE1 in MTX-induced increases in [Ca2þ]i and necrotic cell death in cultured rat cortical neurons. 2. Materials and methods 2.1. Materials Timed pregnant Sprague–Dawley rats (Charles River, Montreal, QC, Canada) were maintained on a 12/12 h light/ dark cycle and had free access to food and water. In conducting this research, the authors adhered to the ‘‘Guide to the Care and Use of Experimental Animals’’ and ‘‘The Ethics of Animal Experimentation’’ published by the Canadian Council on Animal Care. MTX was purchased from Wako

(Richmond, VA). 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and ethidium bromide (EB) were purchased from Sigma– Aldrich (Saint Louis, MO) and dissolved in water. The initial stock solutions of MTX were made in methanol at a concentration of 10 mM and stored at 20  C. Fura-2 AM ester was purchased from Invitrogen (Burlington, ON, Canada) and dissolved in water. All other chemicals were of chemical grade and purchased from Sigma–Aldrich. 2.2. Primary culture of rat cortical neurons Dissociated cultures of cortical neurons were prepared from 17 to 19 day old rat embryos as described previously (Wang et al., 2004) with some modifications. Briefly, cortices were removed into ice cold Hank’s balanced salt solution (HBSS, Ca2þ, Mg2þ free, Invitrogen, Burlington, ON, Canada). The tissues were minced and trypsinized (0.25%, 10 min at 37  C), titrated and plated on poly-D-lysine coated 12 well plates at a density of 106 cells per well. For microscopic studies, cells were plated onto 18 mm round polyD-lysine coated coverslips (Harvard Apparatus, Quebec, Canada). For fluorimetric measurements of [Ca2þ]i, cells were plated onto poly-D-lysine coated 28  12 mm rectangular glass coverslips at a density of 106 cell per coverslip. After plating, cells were cultured in Neurobasal medium supplemented with B-27 and 0.5 mM glutamax (Invitrogen). Cultures were maintained at 37  C in a humidified atmosphere of 5% CO2 and 95% air. The maintenance medium was replaced every 3–4 days and cells were kept for 11–14 days in vitro (DIV) before being used. This procedure has been reported to yield neuronal cultures of more than 95% purity (Lesuisse and Martin, 2002). 2.3. Treatment of cultured neurons with MTX To assess the toxicity of MTX on cultured cortical neurons, cells (11–14 DIV) were incubated in medium containing varying concentrations (0–200 pM) of MTX. After 30 min incubation at 37  C, the treatment medium was replaced with fresh maintenance medium and the cultures returned to the incubator for 18–24 h prior to cell death assays. To test the effect of the NHE inhibitor, EIPA, on MTX-induced neurotoxicity, varying concentrations (1–10 mM) were introduced into the treatment medium 30 min before MTX treatment. EIPA was present until cell viability assays were carried out. 2.4. Fluorimetric measurement of [Ca2þ]i Cells plated on the rectangular coverslips were washed once with maintenance medium and then incubated with 1 mM fura-2 AM in fresh medium for 30 min at 37  C. After the incubation period, cells were washed twice with HEPES buffer (10 mM HEPES, 140 mM NaCl, 25 mM glucose, 5.4 mM KCl, 1.3 mM CaCl2, pH 7.35, osmolarity 310– 320 mosM). The coverslip was then carefully inserted into a fluorimeter cuvette so that it was aligned at 45 to the excitation beam. The cuvette containing the cover slip and 2 ml HEPES buffer was kept at 37  C and under continuous magnetic stirring. A fluorescence ratio of 340/380 nm excitation was obtained by measuring the fluorescence

Y. Wang et al. / Toxicon 54 (2009) 95–102

intensity at 495 nm emission. [Ca2þ]i was calculated according to the following equation, as described previously (Beani et al., 1994):

h

i Ca2þ ¼ Kd ðR  Rmin Þ=ðRmax  RÞðFo =FS Þ

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means  SE. Statistical evaluations were performed with the Student’s t-test. Differences between means were considered significant at p values <0.05. 3. Results

i

Where Kd is 225 nM, R the ratio 340/380 of fluorescence of the indicator, Rmin the ratio in the absence of extracellular Ca2þ, Rmax the ratio in the presence of saturating Ca2þ concentration, and Fo/FS the ratio of 380 nm excitation fluorescence at zero and saturating Ca2þ levels. This procedure yielded a [Ca2þ]i of 10–15 nM under basal conditions, which is consistent with what has been previously reported (Beani et al., 1994). 2.5. Cell viability assay Cell viability was assessed using alamarBlueÔ assay (AccuMed International Inc., Westlake, OH). Briefly, 18–24 h after the initial treatment, alamarBlueÔ dye was added to cultures (10% v/v) and left for 3–4 h in the incubator before an aliquot of the supernatant was transferred to a 96 well titer plate. Fluorescence was measured on a Bio Tek FL600 Microplate Fluorescence Reader (MTX Lab Systems, Inc., Vienna, VA) using an excitation wavelength of 530–560 nm and an emission wavelength of 590 nm. Cell viability was calculated and plotted as a percent of control cells. 2.6. Microscopic imaging of EB uptake Cells plated on 18 mm round coverslips were pre-incubated for 30 min with fresh medium, or medium containing various concentrations of EIPA. After incubation, the coverslips were transferred to a holder containing 1 ml of fresh medium for microscopic imaging. Ethidium bromide (1 mg/ml) was added to the medium prior to image acquisition. Collection of data was performed using a Wave-FX spinning disc confocal microscope (Quorum Technologies, Guelph, Canada) with a laser excitation wavelength of 491 nm and an emission filter of 593 nm at a magnification of 20. Images were collected at 1 min intervals for 31 min, with MTX being added after the first time point. Images collected at time 0 for each group were designated as background and were subtracted from each time point after MTX treatment. 2.7. NHE1 SiRNA tranfection NHE1 SiRNA, its control antibody and related transfection reagents were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). NHE1 SiRNA transfection (100 nm SiRNA per well in 12 well plates) in human embryonic kidney (HEK293) cells was carried out using a protocol provided by the manufacturer. Result of NHE1 knockdown in HEK293 cells was verified by Western blot using a control antibody for NHE1 purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2.8. Data analysis All experiments were carried out in cells from at least four independent cultures and results are presented as

3.1. MTX triggers a rapid increase in [Ca2þ]i and causes cell death in cultured rat cortical neurons Cultured rat cortical neurons kept at 37  C showed a basal [Ca ]i of 15.2  2.1 nM. This basal concentration is in agreement with a previously published report (Beani et al., 1994). Treatment of these neurons with varying concentrations of MTX-induced a dramatic rise in the levels of intracellular Ca2þ, as seen by the increases in both the absolute concentrations and the normalized ratio of 340/380 (Fig. 1). We next examined the uptake of EB into neurons treated with MTX. As shown in Fig. 2A, treatment of neurons with varying concentrations of MTX caused a dramatic increase of EB uptake within 30 min. The uptake could be detected in live cells as early as 5 min after MTX treatment (data not shown). Furthermore, cell viability assay revealed that MTX-induced significant cell death in a concentration dependent manner (Fig. 2B; p < 0.01). It should be noted that the concentration of MTX required to cause both [Ca2þ]i increase and cell death in neurons is much lower than that required for rat aortic smooth muscle cells (unpublished observation), endothelial cells (Wisnoskey et al., 2004) and Chinese hamster ovary (CHO) cells (Cataldi et al., 1999; Morales-Tlalpan and Vaca, 2002). 2þ

3.2. Protection of MTX-induced cell death by extracellular acidosis To test if changes in extracellular pH affected MTXinduced responses in rat cortical neurons, cells were treated with 25 pM MTX in medium with a pH of 6.0, and then left overnight at pH 6.0. As shown in Fig. 3A, exposure of neurons to pH 6.0 medium alone did not affect cell survival (p > 0.05, compared to pH 7.5). Extracellular acidosis significantly diminished MTX-induced cell death (Fig. 3A; p < 0.01). To exclude the possibility that MTX is degraded in acidic conditions, cells were first treated with MTX in normal medium (pH 7.5). After the 30 min treatment, MTX was washed out with normal medium, and then cells were switched to and maintained in low pH (6.0) medium prior to cell death assay. Fig. 3B showed that this strategy was also effective at protecting against MTXinduced cell death, as compared to those cells that were treated with MTX and maintained in pH 7.5 medium throughout the experimental period (Fig. 3). 3.3. Effect of EIPA on MTX-induced [Ca2þ]i increase and cell death in cultured rat cortical neurons Because extracellular acidosis has been shown to inhibit the cell membrane NHE activity (Grinstein et al., 1989; Li et al., 2008; Vornov et al., 1996), we tested the ability of the NHE inhibitor, EIPA to protect against MTX-induced events. As shown in Fig. 4, pretreatment of cells with 5 mM EIPA, significantly attenuated MTX-induced [Ca2þ]i increase

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Time (min) Fig. 1. Intracellular calcium increase induced by various concentrations of MTX in cultured rat cortical neurons. Main graph shows representative traces of normalized fluorescence ratios of 340/380 nm excitation for control and MTX treated neurons. Inset: [Ca2þ]i calculated using the formula shown in Section 2, showing the effect of various concentrations of MTX 25 min after treatment. Values represent mean  SEM from four experiments. **p < 0.01 compared with control.

(p < 0.01). This concentration of EIPA (5 mM) has been shown to selectively inhibit NHE activities in various cell types (Masereel et al., 2003). Fig. 5 showed the effect of EIPA on MTX-induced EB uptake and cell death in cultured rat cortical neurons. Pretreatment of cells with varying concentrations of EIPA dramatically reduced both MTX-induced EB uptake (Fig. 5A) and cell death (Fig. 5B). 3.4. Effect of NHE1 gene knockdown on MTX-induced cell death in cultured rat cortical neurons

transfection. Due to the general low transfection efficiencies in primary neuronal cultures, we chose to use HEK293 cells, which have previously been shown to express NHE1 (Lang et al., 2003). As shown in Fig. 6, NHE1 protein levels were significantly reduced by 100 nM SiRNA transfection. This reduction in NHE1 protein levels was specific because the SiRNA negative control had no effect (Fig. 6A). MTXinduced cell death was significantly attenuated by either 50 or 100 nM NHE1 SiRNA, indicating that NHE1 activation plays an important role in this event (Fig. 6B). 4. Discussion

To confirm that the effects of EIPA and extracellular acidosis on MTX’s actions were caused by specific inhibition of NHE, the NHE1 gene was silenced using SiRNA

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Fig. 2. MTX-induced ethidium bromide (EB) uptake and cell death in cultured rat cortical neurons. (A) Representative photographs of EB uptake 30 min after MTX treatment, along with DIC and merged images captured with a confocal microscope. Magnification: 20; scale bars: 50 mm. (B) MTX-induced total cell death, 18–24 h after MTX treatment, assayed using the alamarBlueÔ assay. Values are mean  SEM from four experiments. **p < 0.01 compared with control.

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Fig. 3. Extracellular acidosis protects MTX-induced cell death in culture rat cortical neurons. Values represent mean  SEM from four experiments. (A) Effect of low pH medium on 25 pM MTX-induced cell death. *p < 0.05, **p < 0.01 compared with their respective controls; ##p < 0.01 compared with MTX treated group at pH 7.5. (B) Effect of different treatment procedures on MTX-induced cell death. pH 7.5–7.5 (C): cells were treated with varying concentrations of MTX in pH 7.5 medium for 30 min, then switched to and maintained in pH 7.5 medium; pH 6.0–7.5 (:): cells were treated with MTX in pH 6.0 medium, then switched to and maintained in pH 7.5 medium; pH 6.0–6.0 (A): cells were treated with MTX in pH 6.0 medium, then switched to and maintained in pH 6.0 medium; pH 7.5–6.0 (-): cells were treated with MTX in pH 7.5 medium, then switched to and maintained in pH 6.0 medium. Values are mean  SEM from four experiments.

a concentration dependent manner in rat cortical neurons. The MTX effects were significantly attenuated by extracellular acidosis, pharmacological inhibition of the cell membrane NHE, or NHE1 gene knockdown by SiRNA, suggesting that the exchanger may play an important role in MTX-induced neurotoxicity. The ability of MTX to trigger Ca2þ influx and necrotic cell death in a variety of tissues has been extensively studied in the past decade (de la Rosa et al., 2007), and it is now clear that the toxin triggers a series of events that involves the opening of the NSCC (Wisnoskey et al., 2004). Although it is known that NSCC is a cation channel, which allows multiple ions including Naþ and Ca2þ to enter the cell, its molecular structure is currently unknown. In addition, the pharmacological characteristics of the NSCC are not well defined, mainly due to the lack of specific inhibitors (Weber

et al., 2000). Studies have suggested that MTX may directly interact with the NSCC to make it permeable to Naþ and Ca2þ, and activation of this cation channel is likely the first step in a series of MTX-triggered events (Wisnoskey et al., 2004). However, it is possible that MTX binds to its own receptor(s), which then regulate the opening of the NSCC and other channels. In fact, previous reports have suggested the existence of a MTX receptor on the cell membrane, although the identity of the receptor remains a mystery (de la Rosa et al., 2007; Lundy et al., 2004). Our finding that the NHE inhibitor, EIPA, almost completely blocked MTXinduced Ca2þ influx and cell death in cultured cortical neurons, suggests that the activation of NHE precedes Ca2þ influx. Therefore, it is possible that the activation of NHE may be the first step in MTX’s actions in these neurons. It remains unclear if MTX directly interacts with NHE, or if it

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Fig. 5. Effect of EIPA on MTX-induced cell death in cultured rat cortical neurons. (A) Representative images of EB uptake 30 min after MTX treatment. Magnification: 20; scale bars: 50 mm. (B) Effect of EIPA on MTX-induced cell death 18–24 h after MTX treatment, assayed with the alamarBlueÔ method. Values are mean  SEM from four experiments. **p < 0.01 compared with control.

activates NHE by other mechanisms. Nonetheless, our results may shed light on the search for the putative MTX receptor(s). Our observation that extracellular acidosis protects cortical neurons against MTX toxicity is somewhat unexpected, because extracellular acidosis is generally believed to be detrimental to the CNS, especially under excitotoxic conditions (Unterberg et al., 2004; Xu and Xiong, 2007). However, it has been suggested that mild extracellular acidosis may serve as an endogenous protective mechanism against various insults, such as brain ischemia,

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hypoxia and stroke (Vornov et al., 1996). Particularly, low extracellular pH has been shown to inhibit NMDA receptor activation, thereby protecting neurons against excitotoxicity (Tombaugh and Sapolsky, 1990). Moreover, acidic conditions have been shown to protect cortical neurons from cell death during recovery from metabolic inhibition, primarily by inhibiting the activity of NHE (Li et al., 2008; Vornov et al., 1996). Therefore, our results are consistent with the hypothesis that extracellular acidosis may protect against MTX-induced toxicity in rat cortical neurons through the inhibition of NHE.

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Fig. 6. Effect of NHE1 SiRNA on MTX-induced cell death in cultured rat cortical neurons. (A) Representative Western blot showing that 100 nM NHE1 SiRNA transfection resulted in significant reduction in NHE1 protein expression in HEK293 cells. (B) MTX-induced cell death was significantly attenuated by NHE1 SiRNA in HEK293 cells. Values are mean  SEM from four experiments. *p < 0.05; **p < 0.01 compared with control; ##p < 0.01 compared with MTX-treated group in control.

Y. Wang et al. / Toxicon 54 (2009) 95–102

Previous reports indicate that the NSCC may be the primary route of Ca2þ entry in most cell types (de la Rosa et al., 2007). However, it is obvious that the NSCC is not the only channel that allows Ca2þ entry after MTX treatment, as it has been reported that voltage sensitive calcium channel (VSCC) blockers can attenuate MTX-induced [Ca2þ]i increases in excitable cells (Kakizaki et al., 2006). Interestingly, it has been suggested that distinct pathways of Ca2þ entry play different roles in Ca2þ responses. Specifically, it has been reported (Sattler et al., 1998) that the source of Ca2þ entry, and not the Ca2þ load, is the main determinant of the neurotoxic potential of this ion. Because Ca2þ entry through VSCCs is generally considered non-toxic to excitable cells (Sattler et al., 1998), it is unlikely that VSCCs contribute significantly to MTX-induced cell death in cultured rat cortical neurons. In addition to its ability to regulate intracellular pH, NHE is tightly coupled to other membrane pumps and transporters such as the Naþ, KþATPase, and the Naþ/Ca2þ exchanger (NCX) (Matsumoto et al., 2004; Putney et al., 2002). Based on our results that EIPA blocked MTX-induced Ca2þ influx and cell death, it is tempting to speculate that Ca2þ may enter the cells through the two membrane channels mentioned above. Indeed, our research with cultured rat aortic endothelial and smooth muscle cells indicated that MTX-induced Ca2þ influx could be blocked by KB-R7943, an inhibitor of the reversed mode of NCX (Frew et al., 2008). Therefore, it is plausible that NHE activation, as an initial response after MTX exposure, causes a large increase in intracellular Naþ. In order to extrude the excessive Naþ, the reverse mode of NCX is activated, resulting in a massive influx of Ca2þ. Whether or not NSCC is also regulated by NHE activation in various cell types including neurons remains to be investigated. MTX has been shown to cause uptake of the vital dye EB into a variety of cell types, presumably through the NSCC (Estacion and Schilling, 2002; Lundy et al., 2004). Ethidium bromide, when bound to the cell nucleus, fluoresces, which is detectable at wavelengths of 600 nm or longer. Under physiological conditions, the intact cell membrane is not permeable to EB. However, when cells are challenged with toxic stimuli, the integrity of the membrane can be compromised, thus allowing EB to enter the cells and bind to the nucleus. Its uptake is initiated by an increase in [Ca2þ]i and typically precedes total cell lysis (Schilling et al., 1999a). Our finding that EB uptake occurs as early as 5 min after MTX treatment, yet cell death can be attenuated even if the pH of the extracellular medium is lowered after the initial 30 min treatment, confirms that EB uptake precedes cell death, and the opening of the EB permeable cytolytic pore may be a reversible process. It can be inferred from these results that MTX neurotoxicity may be alleviated even if the NSCC is already activated. In conclusion, our results suggest that the activation of NHE plays an important role in MTX-induced responses in cultured rat cortical neurons. NHE inhibitors may be effective at antagonizing MTX neurotoxicity. Conflict of interest The authors declare that there are no conflicts of interest.

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