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Antimicrobial Agents and Chemotherapy, February 2008, p. 684-693, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00874-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mefloquine-Induced Disruption of Calcium Homeostasis in Mammalian Cells Is Similar to That Induced by Ionomycin{triangledown}

D. Caridha,1 D. Yourick,2 M. Cabezas,2 L. Wolf,2 T. H. Hudson,1 and G. S. Dow1*

Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, Maryland 20910,1 Division of Neuroscience, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, Maryland 209102

Received 4 July 2007/ Returned for modification 27 August 2007/ Accepted 1 November 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-D-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mefloquine is an antimalarial with utility for chemoprophylaxis and treatment. The drug has been associated with adverse central nervous system (CNS) effects in a dose-related manner (reviewed in reference 14). As a consequence, its continued use in an otherwise healthy population for prophylaxis is controversial. The precise etiology of mefloquine-induced adverse effects is unknown. There is clinical evidence that P glycoprotein polymorphisms are associated with adverse neurological outcomes (1). Numerous putative CNS targets have been proposed (reviewed in reference 12) in either in vitro or ex vivo contexts. Recent studies in our laboratory demonstrated mefloquine induction of brain stem histopathology consistent with direct cellular neurotoxicity in rats (12) and disruption of neuronal calcium homeostasis in vitro (14). The latter effect involves discharge of the endoplasmic reticulum (ER) calcium store and induction of a subsequent influx of calcium into the cell from the extracellular space (14). Initially we suspected that this effect might occur as a consequence of the inhibition of the thapsigargin-sensitive sarcoplasmic reticulum/ER Ca2+-ATPase (SERCA), followed by subsequent triggering of capacitative calcium entry (CCE). However, in subsequent gene expression studies, we observed that mefloquine failed to induce the downstream stress responses typical of thapsigargin-induced ER calcium depletion (13). These observations suggested either that the interaction of mefloquine with the ER calcium pump were transient or that the depletion was mediated via different mechanisms. Alternative mechanisms of disruption could include ryanodine receptors (RyRs), the G protein-coupled inositol phosphate 3 (IP3) signaling pathway, ionophoric or metabotropic glutamate receptors, and/or voltage-gated calcium channels, alone or in combination. Alternatively, a receptor-independent ionophoric mechanism might be responsible. In the present study, we present data that suggest that glutamate receptors are not involved in the disruption of calcium homeostasis by mefloquine and that mefloquine and ionomycin exhibit similar downstream effects on calcium homeostasis in mammalian cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and media. Mefloquine and all of the mefloquine analogs were obtained from the Walter Reed Army Institute of Research Chemical Inventory System. [3H](myo)inositol was purchased from PerkinElmer and Analytical Sciences, MA. N-methyl-D-Aspartic acid (NMDA) antagonists, namely, DL-2-amino-5-phosphonopentanoic acid (AP5), N,N'-1,4-butanediylbisguanidine sulfate (arcaine), and cis-4-[phosphomethyl]-2-piperidinecarboxylic acid (CGS 19755); non-NMDA receptor antagonists, namely, 6,7-dinitroquinoxaline-2,3-dione (DNQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-5-yl)-benzeamine dihydrochloride (GYKI 152466); the group I metabotropic glutamate receptor antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA); DL-2-amino-3-phosphonopropionic acid (AP3); the CCE and receptor-mediated Ca2+ entry antagonist 1-[2-(4-methoxyphenyl)propoxy]ethyl-1-H-imidazole hydrochloride (SKF96365); the L-type (where L stands for long-lasting) Ca2+ channel blocker {alpha}-[3-[[2-(3,4-dimethoxyphenyl)methylamino]propyl]-3,4-dimethoxy-{alpha}-(1-methylethyl)benzeneacetonitrile hydrochloride (verapamil hydrochloride); and other antagonists, such as DL-2-amino-4-phosphonobutyric acid (AP4), were purchased from Tocris Cookson Inc., MO. The SERCA inhibitor thapsigargin, the ryanodine-mediated ER calcium release inhibitors 1-[[[5-(4-nitrophenyl)-2-furanyl]methylene]amino]-2,4-imidazolinedione (dantrolene) and ammoniated ruthenium oxychloride (ruthenium red), and the NMDA receptor antagonist dizocilpine-(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine mealate (MK801) were also purchased from Tocris Cookson Inc. Poly-L-lysine, cytosine B-D-arabinofuranoside, basal Eagle medium with Earle's salts, 48-well plates, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), the phospholipase C (PLC) inhibitor neomycin, and DL-glutamic acid (agonist at the kainate, NMDA, and quisqualate receptors) were purchased from Sigma Aldrich, MO. Penicillin-streptomycin, glutamine, and minimum essential medium were obtained from Invitrogen, while 96-well plates and 4-well slides were obtained from Fisher Scientific Company, GA.

Cell culture. Neuronal primary cell cultures were established using cerebral cortices from embryonic day 15 rats. Neurons were isolated and cultured as previously described (20). Animal care and use was approved by an institutional animal ethics committee in accordance with national guidelines. Research was conducted in compliance with the Animal Welfare Act, other federal statutes, and regulations that relate to animals and experiments involving animals, and principles stated in the Guide for the Care and Use of Laboratory Animals (3). Cells of the macrophage cell line RAW 264.7 were obtained from the American Type Culture Collection and were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, a 1x antibiotic-antimycotic mixture, 4 mM L-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, and 4.5 g/liter glucose. Cells were grown in 75-cm2 flasks and were incubated in a 95% air-5% carbon dioxide (CO2) atmosphere at 37°C. For subcultivation purposes, all but 10 ml of the culture medium was removed from the flask. Cells were scraped from the flask substrate with a cell scraper, transferred to a sterile 15-ml tube, and centrifuged at 3,000 rpm for 10 min at room temperature. Pellets were then resuspended in 10 ml medium, and two 5-ml aliquots of cell suspension were added to new culture vessels, followed by an additional 15 ml of medium. Medium was replaced every 2 to 3 days.

Neurotoxicity and neuroprotection assays. Neuroprotection assays were conducted to determine if particular antagonists protected neurons from toxicity induced by a number of different agonists (methods are outlined in the next paragraph). The pattern of neuroprotection induced by various combinations of antagonists reveals useful information about the mechanism of action of uncharacterized agonists (i.e., mefloquine). In initial experiments (Table 1), mefloquine was an experimental agonist, and the neuroprotective effects of antagonists of the indicated pathways were investigated. The agents and concentrations used are indicated in Table 1. These initial observations suggested the possible involvement of glutamate receptors, so a second series of neuroprotection experiments were performed using different combinations of glutamate agonists and antagonists (Table 2). The agonists employed were mefloquine (an uncharacterized agonist), glutamate (an agonist of all glutamate receptors), kainic acid (an agonist of non-NMDA receptors), and quisqualic acid [an agonist of (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and metabotropic glutamate receptors]. The effects of various glutamate receptor antagonists were tested at the concentrations indicated in Table 2.


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  		TABLE 1. Effects of Ca2+ channel agonists and antagonists on mefloquine toxicitya

 

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  		TABLE 2. Protective effects of glutamate receptor antagonists on the neurotoxicity induced by mefloquine and glutamate receptor agonistsa

 
Prior to the conduction of neuroprotection experiments, the 50% lethal concentrations (LC50s) of various agonists and the inherent toxicities of different concentrations of antagonists were evaluated. Neurotoxicity was assessed by exposing the cultured neurons to graded concentrations of mefloquine and other agonists and assessing viability relative to that of controls using the MTT (thialozyl blue reduction) assay as previously described (14). LC50s were calculated using Prism. The numbers of drug dilutions and replicates are outlined in the appropriate table or figure legend for each experiment. Neuroprotection experiments were conducted as previously described (14). Neurons were exposed to the potential antagonist/neuroprotectant for 5 min and then to the agonist for 20 min, after which viability relative to that of appropriate controls was determined. Controls included no antagonist/agonist, an antagonist alone (negative control), and an agonist alone (positive control). Dimethyl sulfoxide (DMSO) or Locke's solution was used as the diluent for all antagonists/agonists. The final DMSO concentration never exceeded 2.16% (vol/vol) in any individual well, well below the maximum of 8% (vol/vol) tolerated by neurons under these experimental conditions. In the first experiment, concentrations of antagonists/agonists were selected based on precedents in the literature, and their effects on neurons alone were noted. In experiments involving glutamate receptors, each antagonist used was previously shown to induce no neurotoxicity at the concentration selected. Each agonist was used at its approximate LC50 (determined for each compound prior to the experiment), and the test was considered valid if cell killing was in the range 40 to 60%. Each experiment was conducted at least three times. In the first experiment, the effect of the antagonist was considered to be antagonistic, protective, or additively toxic based on its effect on neurons alone and in combination with mefloquine (as noted in Table 1). In the glutamate receptor experiments, an antagonist was considered neuroprotective if cell viability was statistically greater than that with the agonist treatment alone (single factor analysis of variance [ANOVA] and Dunnett's multiple-comparison test). P values of <0.05 were considered significant.

Toxicity and protection assays in macrophages. RAW-264.7 macrophage cells were grown in 75-cm2 tissue culture flasks. For mefloquine and ionomycin cytotoxicity studies, 96-well plates were seeded with 12 x 104 cells 4 to 6 h before the start of the assay. Fifty percent inhibitory concentrations (IC50s) of mefloquine and ionomycin in the presence and absence of different concentrations of antagonists (MgCl2 and DNQX) were determined using Prism. For cytotoxicity experiments, cell medium was removed and replaced with 100 µl Locke's solution. Then, 20 µl of the appropriate concentration of the potential protectant (MgCl2 or DNQX) that yielded the desired concentration of the antagonist was added, and cells were returned to the incubator. Five minutes later, cells were exposed to the agonist for 20 minutes. Then, the drug mixture was removed and 200 µl medium was added to each well. In each assay plate, controls were placed as described under "Neurotoxicity and neuroprotection assays" above. Toxicity and protection were assessed the next morning by using the MTT (thiazolyl blue reduction) assay as previously described (14). The test was considered valid if cell killing in the negative controls (Locke's solution/agonist treatment) was in the 40 to 60% range.

[Ca2+] measurement studies and confocal microscopy. The effects of various antagonists on changes in calcium homeostasis induced by various agonists were evaluated using fluorescence microscopy. The cells (neurons and macrophages) were loaded with the calcium-sensitive dye Fluo-3-AM (5 µM for 1 h), rinsed, and returned to an incubator for 15 min prior to the imaging experiment (14). Changes in cellular calcium homeostasis were monitored using a Bio-Rad Radiance 2000 confocal imaging system. Changes in cytoplasmic calcium were recorded as fluctuations in the emitted fluorescence of Fluo-3-complexed calcium at 530 nm. Sequential image scans of fields containing 5 to 15 cells were used to construct temporal profiles of the effects of the different analogs. To compare the fluorescence levels in different cells (which were often in slightly different focal planes) on different days, readings at each time point were normalized to the first value measured for each neuron. Scans were made at 10-s intervals. Antagonists were added at scan 3 (after 30 s), and agonists were added at scan 18 (after 2.5 min). Cells were monitored for an additional 42 scans (7 min). Controls included no antagonist or agonist (Locke's solution as a negative control) and the agonist alone (positive control). Each control and the combination of agonist and antagonist treatments were tested at least twice each week, and the experiment was repeated for at least three consecutive weeks. Data from all of the replicates were then pooled into a single curve, sketched using Prism, and expressed as percentages of increase in Fluo-3 fluorescence over time. The total numbers of neurons or macrophages from which data were collected are indicated in the respective figure legends.

IP(n) accumulation in neurons and macrophages. Macrophages were grown on 24-well plates, which were seeded with 12 x 105 cells 15 to 16 h before the start of the assay. Nine-day-old primary neurons were cultured as described previously (14). Total inositol phosphate [IP(n)] measurement was performed as previously described (35). Cells were preincubated for 5 min with 100 µM DNQX or Locke's solution as a control, and then plates were swirled and transferred to an incubator for 5 min. Five microliters of an agonist solution (mefloquine, glutamate, or ionomycin in an appropriate concentration to yield the previously measured IC50s) or the control (DMSO) was added. Cells were incubated for 20 min at 37°C with 5% CO2. The preincubation and incubation times were the same as in toxicity studies, so respective results would be comparable. Total IP was eluted using 5 ml of 0.1 M formic acid-1.0 M ammonium formate. The amount of 3H-labeled IP was measured on a model 2200CA United Technologies Packard liquid scintillation analyzer. Each week, there were four to six replicates for each treatment, and the experiments were repeated for three consecutive weeks. The pooled data were analyzed using GraphPad Prism 4 software and are presented as dpm/mg protein and expressed as percentages of the control IP accumulation.

Radioligand binding assays. Radioligand displacement binding assays were performed for specific receptors of interest. These assays were conducted by Novascreen (Hanover, MD). In these assays, the ability of an uncharacterized agonist at various concentrations (mefloquine) to displace a radiolabeled ligand from characterized binding sites of defined targets is evaluated. The results are compared to those of known binders of the target. A potent effect of an uncharacterized agent against a particular target is then interpreted as evidence of an interaction at the known binding sites. Mefloquine was tested in duplicate over a concentration range of 0.000256 to 20 µM against each target (the top dilution was 20 µM, with fivefold dilutions). The targets evaluated were the (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, metabotropic glutamate receptor 5 (mGluR5), NMDA, and IP3 receptors. The ligands displaced were [3H]AMPA, [3H]kainic acid, [3H]MPEP, [3H]GCP 39653, and [3H]IP3 for each of the targets, respectively. Positive controls were AMPA HBr, kainic acid, 2-methyl-6-(phenylethyl)-pyridine (MPEP), NMDA, and D-myo-inositol-1,4,5 (with the compounds present or absent). Assays were performed using standard methods, and IC50s of positive controls were within the range of reference values (Novascreen).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The disruption of neuronal calcium homeostasis by mefloquine could occur as a consequence of effects on one pathway or multiple pathways. In an attempt to identify those involved, we performed neuroprotection experiments, using known antagonists (see Materials and Methods) of important Ca2+ signaling pathways. Our objective was to identify those inhibitors for which we noticed neuroprotection (alleviation of mefloquine toxicity at a nontoxic concentration of the antagonist) or clear antagonism (the toxicity of mefloquine and an antagonist combined was less than the sum the toxicities of both individually). This might indicate the involvement of this pathway in mefloquine toxicity. Several inhibitors of calcium signaling pathways, including dantrolene, thapsigargin, SKF93665, and xestospongin, exhibited an antagonistic effect on mefloquine-induced neurotoxicity (Table 1). Neomycin, an inhibitor of mammalian PLC, exhibited a weak protective effect (Table 1). DNQX, a non-NMDA receptor antagonist, and 12 mM MgCl2 were shown to have a strong neuroprotective effect (Fig. 1; Table 1). The protective effect of the last two agents was relevant to mefloquine-induced changes in calcium homeostasis, since they exhibited a mitigative effect on calcium influx into the cell (Fig. 2).


Figure 1
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  		FIG. 1. DNQX and magnesium at high concentrations protect neurons (A) and magnesium at high concentrations protects macrophages (B) from mefloquine-induced toxicity. Neurons and macrophages were exposed to neuroprotective agents (1.2, 12, and 100 mM MgCl2 and 100 µM DNQX) for 5 min and then with 20 µM mefloquine (MEF) for 20 min. Bars represent means ± standard errors of the means (SEMs) for three pooled experiments containing three replicates per condition (nine experiments per condition). For the data in panel A, single-factor ANOVA was used to determine whether differences in group means existed across the experiment. Differences between the individual group means and values for the mefloquine control were determined using Dunnett's test, with statistical significance (P < 0.05) indicated by *. For data in panel B, an unpaired two-tailed t test was used to evaluate whether there was a statistically significant difference between means in pretreated and nonpretreated groups at 75 µM mefloquine (P < 0.05).

 

Figure 2
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  		FIG. 2. Effects of 100 µM DNQX and 12 mM MgCl2 on calcium homeostasis in neurons treated with 50 µM mefloquine (A and B) and macrophages treated with 100 µM mefloquine (C and D). The effect of 100 µM DNQX and 12 mM MgCl2 on calcium homeostasis was investigated using confocal microscopy. The horizontal axis represents time (in minutes). Cells were loaded with the calcium-sensitive dye Fluo-3 and were scanned at 10-s intervals. The vertical axis represents 530-nm-wavelength fluorescence (F530) normalized to the first value measured for each cell. Arrows show additions of Locke's solution-DMSO, 100 µM DNQX, or 12 mM MgCl2 at scan 3. Mefloquine was added at scan 18. Images from 5 to 8 neurons and 10 to 12 macrophages were collected in a single experiment. Each experiment was repeated three times each session, and data represent pooled data from nine sessions performed during three consecutive weeks. Traces represent the means ± SEMs for 45 to 72 neurons and 90 to 108 macrophages.

 
These initial results suggested that two lines of inquiry were warranted. The protective effect of DNQX and neomycin suggests that mefloquine might activate non-NMDA glutamate receptors. DNQX is a selective non-NMDA receptor antagonist, and neomycin is an inhibitor of PLC, a key component of the IP3 signaling pathway. An alternative hypothesis was that mefloquine induces CCE as a consequence of its discharge of the ER calcium pool. At superphysiological concentrations, magnesium acts as an inhibitor of CCE. While, in theory, NMDA receptors might be implicated in the protective effect of magnesium, we viewed this as unlikely since physiological magnesium concentrations exhibited no protective effect (toxicity or calcium influx [data not shown]).

We first evaluated the possibility that mefloquine might activate non-NMDA glutamate receptors. We compared the neuroprotective effects of various glutamate receptor antagonists on the neurotoxicity induced by mefloquine and known glutamate receptor agonists (L-glutamine, kainic acid, and L-quisqualate, which acts simultaneously at AMPA and group I metabotropic glutamate receptors). We also evaluated the receptor binding affinity of mefloquine to the IP3 and glutamate receptor subtypes and determined whether mefloquine induced phosphatidylinositol hydrolysis. Mefloquine did not induce cellular injury that is similar and consistent with any of the known glutamate agonists (Table 2). CNQX for example, which, like DNQX, is a specific antagonist of ionotropic non-NMDA receptors, did not protect cells from mefloquine-induced toxicity in the same manner as DNQX. Also, high concentrations of L-quisqualic acid are needed (IC50 = 4.5 mM) to cause the same neurotoxic effect as 20 µM mefloquine, yet L-quisqualic acid induced changes in calcium homeostasis at much lower concentrations than mefloquine (data not shown). Mefloquine displayed no affinity for the known binding sites of any of the glutamate receptor subtypes (Table 3).


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  		TABLE 3. Results of radioligand binding assays for mefloquine against multiple targets

 
Taken together, these data seemed to suggest a mechanism of protection by DNQX and magnesium that was (non-NMDA) receptor independent. This was further supported by the lack of isomeric selectivity in the neuroprotection induced by these two agents (Fig. 3). In contrast, mefloquine (together with the positive control, glutamate and histamine), at its LC50 quadrupled IP(n) accumulation in primary neuronal cell cultures. Still, IP(n) accumulation induced by mefloquine was not blocked by DNQX, as one might expect if mefloquine-induced toxicity was receptor mediated (Fig. 4A). Collectively, these data suggested that mefloquine might not be a glutamate receptor agonist and that the protective effects of magnesium and DNQX require further investigation. We therefore turned to our alternative hypothesis—that mefloquine induced CCE as a consequence of an ill-defined upstream effect on the ER calcium store.


Figure 3
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  		FIG. 3. Neuroprotective effect of magnesium and DNQX on the neurotoxic effects of mefloquine isomers. Neurons were exposed to neuroprotective agent for 5 min and then to mefloquine isomers for 20 min. Each week, there were four to six replicates for each plate. Data are pooled from three replicate experiments, done in three consecutive weeks. Bars represent means ± SEMs for a total of 12 to 18 replicates for each experimental condition. Single-factor ANOVA was used to determine whether differences in group means existed across the experiment. Differences between values for the individual group means and the mefloquine control were determined using Dunnett's test. Statistical significance (P < 0.05) is indicated by *. ErythroSR-20, (–)erythro isomer of mefloquine; MG1.2 and MG12, 1.2 and 12 mM MgCl2, respectively; Threo187163 - 50, (+)erythro isomer of mefloquine.

 

Figure 4
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  		FIG. 4. Mefloquine and ionomycin at their IC50s upregulate the IP(n) response in neurons (A), and ionomycin has a similar effect in macrophages (B). DNQX does not affect IP3 upregulation caused by these agonists. Neurons and macrophages were exposed to a neuroprotective agent (100 µM DNQX or the control [DMSO]) for 5 min and then to agonists (20 µM mefloquine [MEF] and 100 µM glutamate [GLU] for neurons and 50 µM mefloquine and 1.8 µM ionomycin [IONO] for macrophages) for 20 min. Each week, there were four to six replicates for each plate. Data are pooled from three replicate experiments, performed in three consecutive weeks. Bars represent means ± SEMs from a total of 12 to 18 replicates for each experimental condition. Single-factor ANOVA was used to determine whether differences in group means existed across the experiment. Differences between the individual group means and the mefloquine control means were determined using Dunnett's test. Statistical significance (P < 0.05) is indicated by *.

 
Prior studies in our laboratory have shown that the disruption of calcium homeostasis in neurons includes two events: an initial release of Ca2+ from internal calcium stores followed by substantial Ca2+ entry from extracellular space (14). This is similar to the effect of Ca2+ ionophores, especially ionomycin (23). If mefloquine acts as an ionophore, one would expect it to exhibit an effect on calcium homeostasis qualitatively similar to that of ionomycin and that this effect would be evident in nonneuronal cell types. Both agents induced sustained dose-dependent elevations in cytoplasmic calcium concentrations in neurons and macrophages (Fig. 5). At lower concentrations, both compounds induced a transient elevation in cytoplasmic calcium. This transient effect correlates with mefloquine-induced store discharge, as demonstrated in our earlier investigations with EGTA-buffered media (14). The effects of both agents were qualitatively similar, although the magnitude of the response to ionomycin was more pronounced (Fig. 5).


Figure 5
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  		FIG. 5. Mefloquine disrupts calcium homeostasis in a dose-dependent manner in neurons (A) and macrophages (B). Ionomycin induces a similar effect in neurons (C) and macrophages (D). The effect of different concentrations (indicated numbers in micromolar units) of mefloquine (MEF) and ionomycin (IONO) in calcium homeostasis was investigated using confocal microscopy. The horizontal axis represents time (in seconds). Cells were loaded with the calcium-sensitive dye Fluo-3 and were scanned at 10-s intervals. The vertical axis represents 530-nm-wavelength fluorescence (F 530) normalized to the first value measured for each cell. Arrows show the addition of Locke's solution-DMSO, ionomycin, or mefloquine at scan 3. For greater clarity, error bars are shown only in the line that represents the highest antagonist concentration, but the errors are all of similar relative sizes for all other agonist concentrations. Images from 5 to 8 neurons and 10 to 12 macrophages were collected in a single experiment. Each experiment was repeated two to three times each week, for three consecutive weeks. Lines represent means ± SEMs for 30 to 72 neurons and 60 to 108 macrophages.

 
In this context, one would expect magnesium, as an inhibitor of CCE, to induce a dose-related mitigative effect on mefloquine- and ionomycin-induced disruption of calcium homeostasis. This was in fact the case in macrophages (Fig. 6). How then to explain the effect of mefloquine on the IP3 signaling pathway and the protective effect of DNQX? In the first instance, ionomycin also upregulated the IP3 pathway (Fig. 4), an effect that may be related to the calcium influx itself, rather than the initial upstream trigger of CCE (see Discussion). DNQX had a mitigative effect on the Ca2+ influx induced by mefloquine in neurons and macrophages and that induced by ionomycin in macrophages (Fig. 2A and C and 7). This effect probably represents a heretofore unobserved off-target inhibitory effect of DNQX on CCE. This is because SKF93665, a CCE inhibitor, made no addition to the mitigative effect of DNQX on the ionomycin-induced disruption of calcium homeostasis in macrophages.


Figure 6
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  		FIG. 6. Effects of different MgCl2 concentrations on calcium homeostasis in macrophages treated with 100 µM mefloquine (A) and 1.8 µM ionomycin (B). The effect of different MgCl2 concentrations on calcium homeostasis was investigated using confocal microscopy. The horizontal axis represents time (in minutes). Cells were loaded with the calcium-sensitive dye Fluo-3 and were scanned at 10-s intervals. The y axis represents 530-nm-wavelength fluorescence (F 530) normalized to the first value measured for each cell. Arrows show additions of Locke's solution; 1.2, 12, and 100 mM MgCl2 at scan 3; and mefloquine or ionomycin at scan 18. For clarity purposes, error bars were shown only on the lines that represent control (Locke's solution) treatments, but the errors are all similar in size. Images from 10 to 12 macrophages were collected in a single experiment. Each experiment was repeated two to three times each week, for three consecutive weeks. Lines represent means ± SEMs for 60 to 108 macrophages.

 

Figure 7
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  		FIG. 7. Effects of 100 µM DNQX, 10 µM SKF93665, and their combination on calcium homeostasis in neurons treated with 1.8 µM ionomycin. The horizontal axis represents time (in minutes). Cells were loaded with the calcium-sensitive dye Fluo-3 and were scanned at 10-s intervals. The y axis represents 530-nm-wavelength fluorescence (F 530) absorbance normalized to the first value measured for each neuron. The left arrow shows the additions of Locke's solution-DMSO, 100 µM DNQX, 10 µM SKF96365 (SKF), and the combination of both at scan 3. The right arrow shows the addition of ionomycin at scan 18. For clarity purposes, error bars were shown only on the line that represents the control treatment, but the errors are all similar in size. Images from 10 to 12 macrophages were collected in a single experiment. Each experiment was repeated two to three times each week, for three consecutive weeks. Lines represent means ± SEMs of 60 to 108 macrophages. Note that the lines representing the DNQX and SKF96365-plus-DNQX treatments overlap.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mefloquine as a non-NMDA receptor antagonist. In earlier studies, we showed that mefloquine disrupted neuronal calcium homeostasis via discharge of the ER store, an event that appeared to trigger subsequent calcium entry into the cell from the external space (14). These events appear to contribute, at least in part, to the acute neurotoxicity of the drug in vitro (14). The precise mechanisms responsible for these events have not been defined. In a subsequent transcriptional study, we showed that mefloquine induced an ER stress response qualitatively dissimilar to that of thapsigargin, an irreversible inhibitor of SERCA (13). This study appeared to indicate either a reversible effect of mefloquine on SERCA or discharge via other mechanisms. In this study, we initially conducted neuroprotection experiments utilizing a variety of antagonists of relevance to pharmacological studies of cellular calcium homeostasis. These initial experiments indicated that magnesium, DNQX, and neomycin protected neurons from injury by mefloquine. These agents are inhibitors of CCE, non-NMDA receptors, and PLC, respectively (9, 17, 27). To us, this suggested the involvement of G protein-linked non-NMDA receptors in the mefloquine-induced disruption of neuronal calcium homeostasis. We reasoned that if this was the case, glutamate receptor antagonists should protect neurons from mefloquine and agonist-induced injury in similar fashions and that mefloquine should induce an IP3 response, displace known glutamate receptor agonists from their binding sites, and not disrupt calcium homeostasis in nonneuronal cells that lack non-NMDA receptors. The fact that three of these events did not occur (Tables 2 and 3; Fig. 4) strongly suggests that non-NMDA receptors are not involved in the disruptive effect of mefloquine on calcium homeostasis (Fig. 8A). The upregulation of the IP3 response is probably secondary to the disruption of calcium homeostasis itself and is addressed below.


Figure 8
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  		FIG. 8. Proposed model for the mechanism of disruption of Ca2+ homeostasis by mefloquine and its similarities with ionomycin. (A) Based on the protective effect of neomycin and DNQX, we initially suspected that mefloquine activated non-NMDA receptors and the G protein-PLC-IP3 pathway. This possibility was ruled out based on receptor-binding studies and the fact that calcium homeostasis was similarly disrupted in nonneuronal cells (macrophages) that do not possess non-NMDA receptors. PIP2, phosphatidyl-inositol biphosphate; IP3R, IP3 receptor. (B) Mefloquine and ionomycin exhibit similar downstream effects on the disruption of cellular calcium homeostasis by releasing Ca2+ from ER stores and, as a result, triggering a CCE response. The relationship between the store discharge and the known and putative ionophoric properties of ionomycin and mefloquine is unclear. (C) Ionomycin acts as a mobile ion carrier. Mefloquine does not have the requisite physiochemical properties to act as a channel former that some ionophores have and probably does not act as a mobile ion carrier. Mefloquine may make membranes more permeable to calcium by virtue of the fact that the drug penetrates and accumulates in biological membranes, thereby disordering their lipid arrays. (D) Other receptors and signaling pathways might be involved in the disruption of Ca2+ homeostasis by mefloquine. Ca2+ entry through voltage-gated channels is most likely excluded as a possible mechanism, since its profile of Ca2+ entry is dissimilar to that of mefloquine. The IP3 upregulation induced by mefloquine (and ionomycin) is likely secondary to the initial disruption of calcium homeostasis. The possibility of the involvement of RyRs and SERCA cannot be excluded based on our data, but their modulation also triggers CCE. R, receptor; G, G protein.

 
Mefloquine and ionomycin exhibit similar downstream effects on cellular calcium homeostasis. Ionomycin is an ionophore, which, based on our data and the literature, appears to have downstream effects on calcium homeostasis that are qualitatively similar to those of mefloquine (the upstream nature of the ionophoric effect of ionomycin is addressed below). At low µM concentrations, ionomycin triggers the discharge of the calcium store, which subsequently triggers CCE (23, 27, 34). These conclusions were based on studies in which ionomycin was shown to antagonize the store discharge induced by histamine (and vice versa) in calcium-free media and in which the subsequent entry of external calcium was blocked by the CCE inhibitor SKF93665 (23). At such concentrations, the contribution of non-CCE-mediated external calcium influx to ionomycin-induced perturbations in calcium homeostasis is negligible (i.e., a direct ionophoric component is lacking) (23). With respect to the ER discharge, we have previously shown a similar effect of mefloquine in relation to the SERCA inhibitor thapsigargin in neurons (14). Given these observations, we wondered if mefloquine might act like ionomycin, at least with respect to its downstream effects. If so, we would expect (i) mefloquine and ionomycin to disrupt calcium homeostasis in qualitatively similar manners in multiple cell types and (ii) the component of disrupted calcium homeostasis attributable to external calcium entry to be inhibitable by SKF93665 and supraphysiological concentrations of magnesium. This was in fact the case for both compounds (Fig. 1 to 2 and 5 to 7). Collectively, these observations suggest that mefloquine and ionomycin exhibit similar downstream effects on calcium homeostasis: discharge of the ER store followed by CCE in multiple cell types (Fig. 8B). We will consider the upstream triggering mechanisms of these effects in the next section.

Mefloquine and ionomycin as ionophores. Mefloquine and ionomycin appear to exhibit similar downstream events on calcium homeostasis. This raises the question of whether mefloquine and ionomycin initiate the same triggering events leading to store discharge. Classically, ionophores are considered to disrupt calcium homeostasis by one of two mechanisms: channel formation or mobile carriage of ions across biological membranes (7). Channel formers create pores in membranes through which ions can diffuse in and out of the cells, while mobile carriers diffuse back and forth across the membranes, transporting cations across a solvent barrier (7). Mobile carrier ionophores can be subdivided into electroneutral or electrogenic ionophores based on whether they function in a charge-dependent or -independent manner (32). Ionomycin is a carboxylic acid ionophore that belongs to the subclass of mobile electroneutral ionophores. Ionomycin forms lipid-soluble cationic complexes with calcium and other cations. This allows it to translocate Ca2+ across membranes in exchange for 2H+ or for another divalent cation (15). However, as discussed earlier, it is unclear to what degree the ionophoric effects of ionomycin actually contribute to its discharge of the internal Ca2+ store. Perhaps an initial ionophoric transient triggers store discharge or the drug translocates across the plasma membrane and preferentially renders the ER membrane more permeable. It is thought that ionomycin does not discharge the store through well-characterized receptor-activated pathways, such as the G protein IP3 cascade or RyRs, but this does not rule out the possibility of the involvement of uncharacterized endogenous signaling cascades (23, 34).

In the context of this lack of clarity regarding the upstream signaling events triggered by ionomycin, what can be concluded regarding the mechanism of action of mefloquine? There are no reports in the literature of which we are aware that have evaluated the possibility that mefloquine induces direct ionophoric effects. However, mefloquine possesses one of the key characteristics of ionophores—it is lipophilic and readily penetrates and accumulates in biological membranes (10, 16, 36). This effect is less marked in other aminoalcohols (quinine) and is not observed with chloroquine (36). Structurally, these effects have been attributed to the planar shape of mefloquine (which allows membrane intercalation), the presence of an ionizable piperidinium nitrogen that may bond with anionic phosphate, H bonding between the hydroxyl group of mefloquine and H bond acceptor groups in the lipid phase, and van der Waals forces between the carbon skeleton of mefloquine and the lipid hydrocarbon chains (16). The result, at least in model membranes, is a significant disordering of lipid side chains and altered temperatures in the transition from the gel phase to the liquid-crystal phase. This effect alone might be sufficient to destabilize membranes and increase their permeability (11, 22). It is also conceivable that, like ionomycin, mefloquine forms complexes with cations via its four-position hydroxyl group and piperidine functional groups and that it acts as a mobile ion carrier (Fig. 8C). However, these possibilities are less intuitively obvious since the carboxylic acid groups of ionomycin are negatively charged, whereas the four-position functional groups of mefloquine are not. The low molecular weight of mefloquine probably precludes the possibility of its acting as a channel former. The general hypothesis that mefloquine is an ionophore should be readily testable in vitro, by monitoring the influx of calcium into Fluo-3 (or another calcium dye)-containing liposomes or vesicles (28). A mefloquine-induced elevation of intraliposomal Ca2+ would confirm that the initiation of the ionophoric effects of the drug may be receptor independent. Such an experiment would significantly advance our understanding of the putative ionophoric effect of mefloquine but would not resolve the issue of the mechanism of store discharge itself.

Possible contributions of endogenous signaling pathways to the mefloquine-induced disruption of calcium homeostasis. In our experimental paradigm, upstream triggers of store discharge could conceivably be either receptor dependent or receptor independent (i.e., ionophoric). It is therefore worth considering the involvement of endogenous signaling pathways. We have definitively excluded the involvement of non-NMDA receptors, the activation of which, a priori, represented the most logical cause of mefloquine-disrupted calcium homeostasis in neurons. However, since we have also observed a similar effect in nonneuronal cells, we must also consider the involvement of other receptors and signaling pathways. Conceivably, the effects of mefloquine could be triggered by the following cellular events: RyR activation and/or the inhibition of SERCA and/or IP3 upregulation followed by CCE or voltage-gated channel activation (6, 19, 21, 29). The possible contributions of these signaling pathways to mefloquine-induced perturbations in calcium homeostasis are discussed below and outlined in Fig. 8D.

Mefloquine was found to induce the IP3 signaling pathway. The nature of the method employed to do this is such that IP3 upregulation may have occurred at any time up to 20 min after mefloquine exposure. However, we suspect that this event occurs secondarily to the disruption of calcium homeostasis by mefloquine. This is because an induction of the IP3 response was also observed after ionomycin treatment. The discharge of the ER store by ionomycin has not been well characterized in mechanistic terms, but it is known that this does not occur via the IP3 signaling pathway (23). Furthermore, IP3 responses secondary to the disruption of calcium homeostasis have been reported (24, 33). Presumably, this contributes to further discharge of the ER store but probably does not act as the initial triggering event. The inhibition of calcium entry by DNQX is surprising in the absence of non-NMDA receptor activation. We interpret this to be due to a previously unreported inhibitory effect on CCE, since the effect was observed in macrophages and DNQX and SKF93665 did not induce additive effects (Fig. 7).

In the case of the RyR receptor, we observed that dantrolene, a specific RyR antagonist, has a weak antagonistic effect on mefloquine-induced toxicity. With toxicity data alone it is difficult to interpret this observation. We cannot rule out the possibility of a non-thapsigargin-like effect of mefloquine on SERCA. The confocal microscopy data and the results of earlier studies are consistent with an effect of mefloquine on this target, although our transcriptional data argue for a reversible, non-thapsigargin-like effect (13). Regardless, CCE is a downstream effect of any of these store-discharging triggers. Voltage-gated channels are present in excitable and endocrine cells, and they are present in both neurons and macrophages (8, 31). Their activity is modulated by phosphorylation/dephosphorylation or by direct control through G proteins (29). In our study, verapamil hydrochloride, a specific inhibitor of the L-type calcium channel, did not antagonize mefloquine toxicity. Some authors report that magnesium acts as an inhibitor of the Ca2+ current through voltage-gated Ca2+ channels. The presence of high concentrations of extracellular and intracellular magnesium ions influences L-type Ca2+ channels, but they activate and deactivate very quickly and this does not match the profile of mefloquine-induced Ca2+ entry (2) (Fig. 8D).

Biological significance of a putative ionophoric effect of mefloquine. Our observations have important biological implications. An ionophoric effect might plausibly be involved in the neurotoxicity of mefloquine in vivo. Mefloquine has been associated with an array of neurological symptoms, including headache, dizziness, vertigo, ataxia, seizures, insomnia, anxiety, and affective disorders (30). These are dose related, since they occur at a higher frequency at doses used for the treatment of malaria (26). Recently, we observed that in rats mefloquine induces dose-related ataxia and histopathologic injuries to the brain stem nuclei that control proprioception (12). The threshold dose for this effect was approximately equivalent to the rat equivalent of the human treatment dose (12). We speculated that these effects represent a correlate of the ataxia and dizziness observed in humans. It is plausible that these adverse events in rats may involve an ionophoric effect of mefloquine. First, mefloquine accumulates in the CNS to levels (90 µM equivalent) that exceed the threshold concentration of mefloquine required for the induction of an ionophoric effect (Table 4). Second, given the affinity of mefloquine for lipids, it is likely that much of the mefloquine in the CNS is partitioned into cell membranes (10, 16, 36). Finally, the nature of the histopathological changes observed suggests that mefloquine causes neuronal degeneration consistent with the necrotic effect in rats in vivo (12). This is consistent with, but not evidence of, an ionophoric effect. One could definitively demonstrate the in vivo relevance of this mechanism by coadministering magnesium intracerebroventricularly prior to mefloquine administration in order to determine whether mefloquine-induced brain stem histopathology and ataxia could be attenuated. If so, it would suggest that cellular events that we have observed in vitro may also occur in vivo in the context of a clinically relevant neuronal injury.


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  		TABLE 4. Accumulation of mefloquine in the brain and plasma

 
Conclusion. In this study, we have shown that the disruption of calcium homeostasis in neurons by mefloquine is not due to an interaction of the drug with specific neuronal receptors. In fact, mefloquine induces perturbations in calcium homeostasis in multiple cell types. This appears to be mediated via the discharge of the internal store, followed by CCE, and is qualitatively similar to the effects of the ionophore ionomycin. Mefloquine has some of the requisite physiochemical properties of an ionophore and has high affinity for biological membranes. Therefore, we suspect that mefloquine may act as an ionophore in mammalian cells. We are in the process of conducting the experiments necessary to test this hypothesis in vitro and assess its possible relevance in vivo.


   ACKNOWLEDGMENTS
 
The manuscript was reviewed by WRAIR and USAMRMC and there is no objection to its publication or dissemination.

The opinions expressed herein are those of the authors and do not reflect the views or opinions of the Department of the Army or the Department of Defense.

This work was conducted under the auspices of the Military Infectious Diseases Research Program (proposal A50030_03_WR_OC).


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910. Phone: (301) 319-9009. Fax: (301) 319-9449. E-mail: geoffrey.dow{at}na.amedd.army.mil Back

{triangledown} Published ahead of print on 12 November 2007. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Antimicrobial Agents and Chemotherapy, February 2008, p. 684-693, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00874-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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