effects of 50 hz electromagnetic fields on voltage-gated ca2+ channels and their role in modulation...

Cell Calcium 35 (2004) 307–315

Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channelsand their role in modulation of neuroendocrine cell proliferation and death

Claudio Grassia,∗, Marcello D’Ascenzoa, Angela Torsellob, Giovanni Martinottia,Federica Wolfb, Achille Cittadinib, Gian Battista Azzenaa

a Institute of Human Physiology, Medical School, Catholic University “S. Cuore”, Largo F. Vito 1, 00168 Rome, Italyb Institute of General Pathology, Medical School, Catholic University “S. Cuore”, Largo F. Vito 1, 00168 Rome, Italy

Received 24 May 2003; received in revised form 7 August 2003; accepted 7 September 2003

Abstract

Possible correlation between the effects of electromagnetic fields (EFs) on voltage-gated Ca2+ channels, cell proliferation and apoptosiswas investigated in neural and neuroendocrine cells. Exposure to 50 Hz EFs significantly enhanced proliferation in human neuroblas-toma IMR32 (+40%) and rat pituitary GH3 cells (+38%). In IMR32 cells EF stimulation also inhibited puromycin- and H2O2-inducedapoptosis (−22 and−33%, respectively). EF effects on proliferation and apoptosis were counteracted by Ca2+ channel blockade. Inwhole-cell patch-clamp experiments 24–72 h exposure to EFs increased macroscopic Ba2+-current density in both GH3 (+67%) andIMR32 cells (+40%). Single-channel recordings showed that gating of L and N channels was instead unaffected, thus suggesting that theobserved enhancement of current density was due to increased number of voltage-gated Ca2+ channels. Western blot analysis of plasmamembrane-enriched microsomal fractions of GH3 and IMR32 cells confirmed enhanced expression of Ca2+ channel subunit�1 followingexposure to EFs. These data provide the first direct evidence that EFs enhance the expression of voltage-gated Ca2+ channels on plasmamembrane of the exposed cells. The consequent increase in Ca2+ influx is likely responsible for the EF-induced modulation of neuronalcell proliferation and apoptosis.© 2003 Elsevier Ltd. All rights reserved.

Keywords: Extremely low-frequency electromagnetic fields; Calcium channels; Cell proliferation; Apoptosis; Channel expression; Neuroblastoma; Ratpituitary cells

1. Introduction

Numerous experimental findings suggest that exposure toextremely low-frequency (ELF) electromagnetic fields (EFs)affects various cell functions via actions exerted on intracel-lular and membrane proteins, including ion channels, mem-brane receptors and enzymes[1,2]. Epidemiological studieson populations living or working within ELFEFs have alsorevealed possible correlation between exposure to thesefields and neoplastic diseases[1,2]. However, the results ofthese studies are notoriously difficult to interpret due to themany overlapping and potentially confounding variablesassociated with this exposure and the multifactorial natureof the diseases observed. Data from experimental studiesare also not univocal. However, the most widely held viewis that ELFEFs may promote carcinogenesis by facilitating

∗ Corresponding author. Tel.:+39-06-3015-4966;fax: +39-06-3015-4665.

E-mail address: [email protected] (C. Grassi).

or favoring the proliferation of genetically altered cells[2].Several in vitro studies have suggested that ELFEFs can af-fect DNA synthesis, RNA transcription and cell proliferationalthough the mechanism(s) underlying these effects havenot been fully defined[3–5]. Alterations in Ca2+ home-ostasis are thought to be involved[5–8] and attempts havebeen made to identify the possible effects of electromag-netic radiation on Ca2+ influx through the cell membranein non-excitable cells[9]. EF exposure reportedly modifiedintracellular Ca2+ levels in rat thymic lymphocytes, humanT-lymphocytes, Jurkat cells and rat pituitary cells[10–13].However, no clear experimental evidence is available yet onthe mechanism through which an increase in the intracel-lular Ca2+ concentrations is obtained. Data have been alsoreported showing that ELFEFs up to 2.0 mT in intensity hadno effect on Ca2+ influx in bovine chromaffin cells[14].

The discrepancies in these findings prompted us to in-vestigate the action of EFs on voltage-gated Ca2+ channels(VGCCs) and the possible correlation between EF-inducedeffects on VGCCs, proliferation and apoptosis, in neuronal

0143-4160/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.ceca.2003.09.001

308 C. Grassi et al. / Cell Calcium 35 (2004) 307–315

and neuroendocrine cells. Exposure of these cells to 50 HzEFs enhanced proliferation and inhibited puromycin- andH2O2-induced apoptosis, both effects being counteracted bythe blockade of VGCCs. Novel experimental evidence isalso provided that ELFEFs increased Ca2+ current densitywithout affecting Ca2+ channel gating. Enhanced expressionof VGCCs, also confirmed by Western blot experiments, istherefore the suggested mechanism underlying the calciuminflux increase responsible for ELFEF effects on neuronaland neuroendocrine cell turnover.

2. Materials and methods

2.1. Cell cultures

Human neuroblastoma IMR32 and rat pituitary GH3 cellswere cultured at 37◦C in an atmosphere of 5% CO2 inair. The former were grown in minimum essential medium(Biochrom KG, Berlin, Germany) supplemented with 10%heat-inactivated fetal bovine serum (HyClone Lab. Inc., Lo-gan, UT), the latter in Ham’s F-10 medium (Biochrom) with17.5% horse serum plus 2.5% fetal calf serum (FCS, Hy-Clone). Both media also contained 100 IU/ml penicillin and100�g/ml streptomycin (Gibco, Grand Island, NY). In somepreparations, IMR32 cell differentiation was induced with1 mM dibutyryl cAMP and 2.5�M 5-bromodeoxyuridine(Sigma Chemical Co., St. Louis, MO) added to the culturemedium three times a week from the day after plating.

2.2. EF exposure

EF exposure was produced with two solenoids, eachcapable of generating EFs characterized by a sinusoidalwaveform with amplitudes of 5–1000�T and frequenciesof 1–100 Hz. For acute exposure during patch-clamp ex-periments, a small solenoid was placed around the 35-mmPetri dish in the inverted microscope. For longer exposures(1–4 days), a much larger solenoid was used inside a CO2incubator, where it was positioned around a Plexiglas cylin-der (20 cm diameter) containing test-cell culture flasks andPetri dishes. This device was supplied by a power gener-ator, and EF frequency and amplitude were monitored byan EF sensor connected to a digital multimeter and oscillo-scope. Control cells were usually grown in a different areaof the same incubator; EF intensity in this area was consis-tently found to be less than 1% of that inside the solenoid.Occasionally, control cells were grown in a separate CO2incubator. Results obtained under these two control condi-tions were not significantly different, and therefore the datawere pooled. Thermometric probes (Homeothermic ControlUnit, Harvard Apparatus Ltd., Edenbridge, UK; 0.1◦C ac-curacy) placed in cell culture dishes inside and outside theMF generator revealed no significant temperature differ-ences between culture media of ELFEF-exposed and controlcells.

2.3. Cell proliferation and apoptosis

Cell proliferation was quantified by means of light micro-scopic cell counts 24, 48, and 72 h after plating. Trypan bluewas used to evaluate cell viability. In some experiments, cellnumbers were also assessed with automated counts (CoulterZ series cell counter, Coulter Corporation, FL).

Apoptosis was evaluated under basal conditions and af-ter cell treatment with the apoptotic agents puromycin andH2O2. Quantitative determination of apoptosis was per-formed by fluorescent microscopy on acridine-orange-stainedcells by scoring for morphological features of nuclear py-knosis and chromatin condensation. Apoptosis was inducedby treating IMR32 cells with either the protein synthesisinhibitor puromycin (10�g/ml for 12 h) or H2O2 (1 mM for30 min). The last treatment induces severe oxygen radicaldamage triggering the programmed cell death. After treat-ment with the apoptotic agents, cells were harvested andwashed in PBS, fixed in cold 4% formaldehyde-ethanol andstored at 4◦C overnight. After three washes in de-ionizedwater, cells were stained with 10�g/ml acridine orange(Sigma). Nuclear pyknosis and chromatin condensationwere scored by a fluorescent microscope Nikon-Eclipse-600(Nikon Corporation, Tokyo, Japan). At least 300 cells werecounted for each condition and double blind examinationwas performed.

2.4. Patch-clamp recordings

Macroscopic and unitary currents of VGCCs wererecorded using the patch-clamp technique in whole-celland cell-attached configurations, respectively[15] (for de-tails, see[16,17]). An Axopatch 200B amplifier (AxonInstruments, Union City, CA) was used; stimulation anddata acquisition were performed with the Digidata 1200series interface and pCLAMP 6.0.3 software (Axon In-struments). Electrodes were pulled from borosilicateglass capillaries; filled with the standard internal solu-tion, their resistance was 3–5 M� for whole-cell record-ings and 4–9 M� for cell-attached recordings. Macro-scopic and single-channel currents were filtered at 5and 2 kHz, respectively. Before recordings, the culturemedium was replaced with Tyrode’s solution containing(mM): 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 glucoseand 10 4-(2-hydroxyethyl)-piperazineethanesulphonic acid(HEPES); pH was adjusted to 7.4 with NaOH.

For whole-cell recordings the external solution was (mM):125 NaCl, 10 BaCl2, 1 MgCl2, 10 HEPES and, to blockNa+-currents, 0.0001 tetrodotoxin (TTX); pH was adjustedto 7.3 with NaOH. The standard internal solution con-tained (mM): 110 CsCl, 10 tetraethylammonium chloride(TEA-Cl), 2 MgCl2, 10 ethylenebis(oxonitrilo)tetraacetate(EGTA), 8 glucose, 10 HEPES, and, to minimize currentrun-down during experiments, 4.0 adenosine 5′-triphosphate(ATP) magnesium salt, 0.25 adenosine 3′,5′-cyclic monophos-phate (cAMP) sodium salt and 4.0 phosphocreatine dis-

C. Grassi et al. / Cell Calcium 35 (2004) 307–315 309

odium salt; pH was adjusted to 7.3 with CsOH. Cellmembranes were depolarized every 10 s (pulse duration:100–140 ms) at voltages ranging from−40 to +50 mVfrom the holding potential (Vh) of −90 mV. Current density(pA/pF) was estimated by dividing current amplitude bymembrane capacitance, measured by theCslow compensa-tion setting of the patch-clamp amplifier.

The cell-attached configuration was used to record unitaryactivity of L- and N-type Ca2+-channels in GH3 and IMR32cells, respectively. The pipette solution contained (mM): 100BaCl2, 10 TEA-Cl, 1 MgCl2, 10 Na-HEPES, 0.0003 TTX(pH adjusted to 7.3 with TEAOH). Membrane potential waszeroed by perfusing the cell with control solution containing(mM): 135 KAsp, 1 MgCl2, 10 HEPES, 5 EGTA, and 0.0003TTX (pH adjusted to 7.3 with KOH). In experiments per-formed to study N channels, the internal solution also con-tained 5�M nifedipine to block L channels; when L-channelactivity was investigated, P/Q and N channels were blockedwith 10�M �-conotoxin-MVIIC (CTx-MVIIC) and 5�M(−)-Bay K 8644 was added to the pipette solution.

Current traces were acquired at 10 kHz. Single-channelactivity was recorded during 120–500 ms depolarizingpulses ranging from−10 to+10 mV fromVh = −40 mV forL channels and from+10 to +30 mV from Vh = −80 mVfor N channels. Data were analyzed with TAC and TAC-FIT software (version 3.04; Bruxton Corporation, Seattle,WA). Fast capacitative transients were minimized on-lineby patch-clamp analogue compensation. Uncorrected ca-pacitative currents were eliminated by averaging sweepswith no channel activity (nulls) and subtracting them fromeach active sweep. Event detection was performed with the50% threshold detection method, and each transition wasvisually inspected before being accepted.

Patches containing unitary openings were used to studythe effects of ELFEFs on the single-channel open proba-bility (Po), mean open time and mean closed time. ThePowas evaluated after exclusion of first and last closures. His-tograms representing open and closed times were plotted onsquare root-log coordinates and constructed as previouslydescribed[16]. Data were not corrected for missed events,and the distributions of open and closed times were fittedby the sum of decaying exponential. For open time distribu-tions, unitary data events from patches with more than onechannel were included in the analysis to increase the num-ber of studied events. The mean amplitude of the unitarycurrent was determined by fitting the amplitude histogramswith a Gaussian distribution. Unitary conductance was eval-uated by linear regression of mean unitary currents recordedat voltages ranging from−10 to+10 mV for L channels andfrom +10 to+30 mV for N channels.

2.5. Western blotting

ELFEF-induced changes in the expression of theCa2+-channel subunit�1 was investigated by Westernblotting. Cell membrane-enriched microsomal fractions of

control and exposed GH3 and IMR32 cells were preparedas described by Gerlach et al.[18] with minor modifica-tions. Cells (5× 107 for each condition) were harvested onice and washed three times by centrifugation (1200 rpm for5 min) with PBS. Pellets were resuspended for 10 min inhypotonic lysis buffer (10 mM KCl, 1.5 mM MgCl2, 2 mMPMSF, 10 mM Tris–HCl, pH 7.4) (4◦C) at a concentrationof approximately 5×107 ml−1. Swollen cells were rupturedin a potter (50 or more strokes) and the homogenate cen-trifuged (3000 and 14,000 rpm for 10 min) to remove celldebris. A cell membrane-enriched microsomal fraction wasseparated by ultracentrifugation (50,000 rpm, 60 min, 4◦C).Membranes were solubilized with 2% (w/v) sodium dode-cyl sulfate, 50 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid, 10% (w/v) sucrose, 10 mM Tris–HCl, pH8.0, heated in a boiling water bath for 5 min. Membraneprotein content was determined by Biorad protein assay(Biorad Laboratories GmbH, Munich, Germany), and thefraction was stored at−80◦C. Western blot analysis wasperformed on membrane proteins that had been separatedby SDS–PAGE (12%) and transferred to immobilion-Pmembranes (Millipore, Bedford, MA) at 100 V for 1 h. Thesame quantity of protein (100�g) was charged in the gel foreach sample of control and ELF-exposed cells. Polyclonalantibody to the pan-�1 subunit of Ca2+ channels (AlomoneLabs, Jerusalem, Israel) was applied (1:200 dilution). Thisantibody recognizes�1 subunit of all the high-voltageactivated (HVA) Ca2+ channels. The housekeeping pro-tein aquaporin AQP-3 was detected as internal control bypolyclonal antibody AQP-3 (C18) at 1:200 dilution (SantaCruz Biotechnology, Santa Cruz, CA, USA). An enhancedchemiluminescence kit was used for Western blot detection(Amersham Pharmacia Biotech, Freiburg, Germany).

2.6. Drugs and solutions

The following compounds were used: puromycin andH2O2 (Sigma), CTx-MVIIC and ω-conotoxin-GVIA(CTx-GVIA Alomone Labs). Nifedipine (Sigma) was di-luted before each experiment from 1 mM stock solution inethanol, which was stored in the dark at 4◦C. In experimentsin which nifedipine was added to cell cultures, flasks werelight-protected, and ethanol was added to control cells (nottreated with nifedipine) at the same concentration presentin cell cultures treated with nifedipine.

Data are presented as means± S.E.M. Student’st-testwas used for statistical analysis, andP-values less than 0.05were considered significant.

3. Results

Most of the parameters considered in this study (cell pro-liferation, macroscopic and unitary Ca2+ currents and Ca2+channel expression) were initially investigated in both hu-man neuroblastoma IMR32 and rat pituitary GH3 cells. In


Fig. 1. Effect of EF stimulation (50 Hz, 1 mT) on proliferation of humanneuroblastoma IMR32 (A) and rat pituitary GH3 (B) cells. Black columns:controls; white columns: ELFEF-exposed cells. (C) Changes in IMR32cell proliferation observed 24, 48 and 72 h after plating. Circles: controls;squares: ELFEF stimulated cells.

each case, the effects of EFLEF exposure proved to be sim-ilar in the two cell lines and further experiments aimed atclarifying the observed effects were usually conducted onIMR32 cells alone.

3.1. Exposure to ELFEF increases cell proliferation

In IMR32 and GH3 cells exposed to 50 Hz EFs at 1.0 mTfor 24–72 h after plating, light-microscopy cell counts re-vealed significantly enhanced proliferation with mean in-creases of 39.9 ± 5.4% (n = 14, P < 0.05) in IMR32 and38.0 ± 7.1% (n = 12, P < 0.05) in GH3 cells (Fig. 1). Insubsequent experiments conducted on IMR32 cells alone,similar increases were documented by automated cell counts(36.0 ± 9.1%; n = 8). The magnitude of the increase wasvery similar at 24, 48 and 72 h (Fig. 1C), suggesting that itwas not due to acceleration of the proliferation rate. The ob-served effects were intensity-dependent with cell count in-creases of 18.1± 7.4% (n = 12) in IMR32 cells exposed to500�T ELFEF and 23.0 ± 4.9% (n = 5) at 750�T. There-fore, in most of the experiments described below, we used a1-mT EF, which produced the greatest effects observed. Thepresence of 50 Hz EFs ranging from 0.5 to 1.0 mT producedno significant thermal changes: temperatures of cell culturedishes inside the MF generator and those in other areas ofthe CO2 incubator remained stable at 37.0 ± 0.1◦C.

To identify the possible role of Ca2+ influx throughVGCCs in the proliferative effects of ELFEFs, we repeatedexperiments in the presence of Cd2+ (15–50�M), whichblocks all types of HVA Ca2+ channels. Cd2+, at concentra-

tions of 25–50�M, inhibited the ELFEF-induced increasein cell proliferation but also exerted some cytotoxic ef-fects, as shown by 11–23% reductions in the number ofviable cells in non-EF-exposed cultures. Lower Cd2+ con-centrations (15�M) exerted no significant effects on cellviability and since this concentration was sufficient to block95.4 ± 0.3% (n = 6) of HVA currents in our models (datafrom patch-clamp experiments described below), it wasused for Ca2+ channel blockade in subsequent experiments.

In the presence of 15�M Cd2+, EF-exposed cell countswere not significantly different from controls in both GH3(n = 7) and IMR32 cells (n = 3), indicating that the ef-fects of ELFEFs on cell proliferation are abolished by block-ade of HVA Ca2+ channels. When dihydropiridine-sensitive(L-type) Ca2+ channels were selectively blocked by 5�Mnifedipine, some increase in proliferation was still observedin exposed cells, but it was significantly smaller than thatseen without any type of Ca2+ channel blockade (19.8 ±6.0% versus 39.9 ± 5.4% in IMR32 cells,P < 0.05; and15.4 ± 4.2% versus 38.0 ± 7.1% in GH3 cells,P < 0.01).

3.2. ELFEF stimulation reduces puromycin- andH2O2-induced apoptosis

In another group of experiments we investigated the ef-fects of ELFEFs on the rate of programmed cell death inIMR32 cells. Morphological evidence of nuclear fragmen-tation was found in 3.3± 0.2% of the ELFEF-exposed cellsversus 4.4 ± 0.4% of non-exposed controls (n = 5). Giventhe low rates observed in both groups, the reduction in thepercentage of apoptotic cells found in EF-exposed cells wasconsidered potentially unreliable, and studies were thus re-peated after induction of apoptosis with the protein synthesisinhibitor puromycin or the pro-oxidant agent H2O2 (Fig. 2).Drug concentrations and treatment duration were adjustedto induce apoptosis in approximately 40–60% of the totalcell number. After 12-h treatment with 10�g/ml puromycin,nuclear fragmentation was found in 61.0 ± 5.8% (n = 7)of control cells. In cells exposed to 1 mT EF at 50 Hz for72 h (60 h before and 12 h after addition of puromycin), thepercentage of apoptotic cells was only 48.1 ± 5.7%, i.e.22.2 ± 3.5% lower than control values (n = 7, P < 0.05).Smaller reductions (11.5 ± 1.8%) were seen with 750�Tfield exposure, and no significant effects were produced by500�T ELFEFs. Comparable effects were seen when apop-tosis was induced with 1 mM H2O2 (Fig. 2B). Treatment for30 min caused nuclear fragmentation in 39.3±1.1% of con-trol cells and 26.3±1.8% of those exposed for 72 h to 1 mTELFEFs, indicating a 33.1± 2.7% decrease in programmedcell death (n = 3, P < 0.005).

To investigate the possible role of calcium influx inthese effects, experiments were repeated in the presenceof VGCC-blocking agents. L channel blockade with 5�Mnifedipine almost completely abolished the anti-apoptoticeffect of ELFEFs in IMR32 cells treated with puromycin(Fig. 2A). In nifedipine-treated control cells, the percentage


Fig. 2. Antiapoptotic action of EFs and its dependence on voltage-gatedCa2+-channels. Apoptosis was induced with 10�g/ml puromycin for12 h (A) or 1 mM H2O2 for 30 min (B). EF-induced reductions in thepercentage of apoptotic cells are shown in the absence of VGCC blockadeand after the blockade of L channels (5�M nifedipine), N channels (3�MCTx-GVIA) and all HVA Ca2+-channels (15�M Cd2+). Black columns:percentage of apoptotic cells under control conditions (non-EF-exposure)normalized to 100%; white columns: apoptosis in ELFEF-exposed cellsexpressed as percentage of controls.∗P < 0.05; ∗∗P < 0.005.

of apoptotic cells was 60.2 ± 2.1% (n = 5), which was notsignificantly higher than that observed (57.2±2.4%,n = 5)in nifedipine-treated cells exposed to EFs. In contrast, theinhibitory effects of ELFEF exposure on apoptosis were stillevident (50.7±5.7% versus 60.0±0.7% with a 15.6±3.1%reduction) following selective N-channel blockade with3.0�M CTx-GVIA (Fig. 2A). Comparable results emergedwhen apoptosis was induced by H2O2 (Fig. 2B). UnderL-channel blockade (nifedipine 5�M) the rates of apop-tosis in control and EF-exposed cells (43.7 ± 1.8% versus39.3± 2.2% n = 3) were not significantly different; similarresults were seen when all HVA Ca2+ channels were blockedby 15�M Cd2+ (43.2 ± 2.1% versus 39.8 ± 1.9%, n = 3).

In preliminary experiments, cell treatment with nifedip-ine alone (i.e. without exposure to any pro-apoptotic or EFstimulation) had no significant effect on the percentage ofapoptotic cells (4.8±0.6% in nifedipine-treated cells versus4.4 ± 0.4% in controls;n = 5).

3.3. Effects of ELFEFs on macroscopic currents throughvoltage-gated Ca2+ channels

The data reported above suggest that the proliferative andantiapoptotic effects of ELFEFs in neuronal and neuroen-

docrine cells are related to the function of the VGCCs. Tofurther investigate this possibility, experiments were firstconducted to determine whether acute ELFEF stimulationdirectly modifies Ca2+ flux through the cell membranes ofIMR32 and GH3 cells. This study performed by using thepatch-clamp technique in whole-cell configuration and it waslimited to the first 5–10 min of EF exposure since longer cur-rent recordings often showed appreciable signs of rundown.At intensities of 200–1000�T and frequencies ranging from16 to 50 Hz, acute ELFEF exposure had no significant effecton the amplitude or kinetics of macroscopic Ba2+ currentsin either cell line.

Next we evaluated the effects of more prolonged ELFEFstimulation like that used in the experiments on cell prolifer-ation and apoptosis. In these experiments, macroscopic Ba2+current densities (seeSection 2) were measured in controland ELFEF-exposed cells. In GH3 cells 3–4 days of ELFEFexposure increased current density by 67.2% (36± 6 pA/pFversus 22± 3 pA/pF in cells grown for the same amount oftime under control conditions) (n = 20, P < 0.05; Fig. 3).Shorter exposure (24 h) enhanced Ba2+ current density byonly 21.9%. Similar results were observed in IMR32 cells.When these cells were treated with differentiating agentswhich promote Ca2+ channel expression (1 mM dibutyrylcAMP and 2.5�M 5-bromodeoxyuridine), ELFEF exposureproduced a 40.4% increase in current density (23±3 pA/pFversus 17±2 pA/pF in controls,n = 106,P < 0.05). In cellsgrown without these agents, Ba2+ current densities weremarkedly lower than those observed in the previous exper-iments, but the increase produced by ELFEF exposure wasmuch more substantial (85.2%,n = 37, P < 0.05). Cellcapacitance of both GH3 and IMR32 cells was not signifi-cantly modified by ELFEF exposure. In particular, the meanvalue of cell capacitance was 7.3±0.9 pF in ELF-stimulatedGH3 cells and 6.5 ± 0.6 pF in controls (P > 0.05). Simi-larly, capacitance of IMR32 cells exposed to ELFEFs was5.7±0.2 pF versus 6.2±0.2 pF of controls. These data showthat no significant change in the cell dimension occurs fol-lowing ELFEF stimulation.

GH3 and IMR32 cells express different HVA Ca2+chan-nels, the L-type being largely prevalent in GH3 cells[19]while IMR32 cells mainly express N channels[20]. The sim-ilar increase in current density observed in the two studiedcell lines suggests that ELFEF action is not specifically ex-erted on one of these two channels. To further verify this hy-pothesis we measured current density in ELF-exposed andcontrol cells before and after application of 5�M nifedip-ine. In GH3 cells the nifedipine-sensitive (L-type) currentwas 61.1 ± 4.0% of the total HVA current and L-currentdensity in ELFEF-exposed cells was 52.3% larger than incontrols. In IMR32 cells, the nifedipine-resistant current wasalmost completely (>90%) flowing through N-type channels[17] and its density was increased by 39.1% following ELFexposure. These data indicate that current densities of L-and N-type channels are similarly enhanced by exposure toELFEF.


Fig. 3. Effects of EF stimulation on Ca2+-channels. A and B show rep-resentative traces of macroscopic Ba2+ current density in control andEF-exposed GH3 cells, respectively. Mean current density increases in-duced by ELFEFs in GH3 (C) and IMR32 cells (D). In the latter cells,changes in current density were measured in the presence (diff.) andabsence (no diff.) of differentiating agents that promote Ca2+-channelexpression; in (C) and (D) black columns: controls; white columns:ELFEF-exposed cells. (E) Increased expression of the VGCC subunit�1

(upper panel) detected in ELFEF-exposed GH3 and IMR32 cells by West-ern blot; aquaporin AQP-3 was used as internal control (lower panel).

3.4. Single-channel properties of N- and L-type Ca2+channels are unaffected by ELFEFs

The increased current density described above indicatesthat 50 Hz EF stimulation augments calcium influx through

Table 1Biophysical characteristics of the unitary L- and N-type currents in control cells and after ELFEF exposure

Current property GH3 (L-type) IMR32 (N-type)

Control ELF Control ELF

Unitary currenta −1.17 ± 0.02 pA −1.16 ± 0.03 pA −1.09 ± 0.03 pA −1.10 ± 0.03 pASlopeγ 21.8 ± 0.6 pS 21.6± 2.0 pS 19.0± 2.3 pS 18.9± 0.5 pSMean open timea 5.60 ± 0.20 ms 5.72± 0.20 ms 1.24± 0.20 ms 1.18± 0.20 ms〈τo〉a 5.08 ms 5.22 ms 1.10 ms 1.06 msMean closed timea 16.70± 1.10 ms 15.05± 1.40 ms 9.20± 0.70 ms 8.71± 0.50 ms〈τc〉a 15.65 ms 13.85 ms 8.33 ms 7.26 msOpen probabilitya 0.24 ± 0.02 0.28± 0.03 0.11± 0.01 0.11± 0.01

a Measured at+10 mV in GH3 cells and at+20 mV in IMR32 cells.

VGCCs. To determine whether this effect was due to anincrease in the number of Ca2+ channels and/or to changesin their functional properties, we made single-channelpatch-clamp recordings in the cell-attached configuration.L channels were studied in GH3 cells in the presence ofCTx-MVIIC and N channels in IMR32 cells with 5�Mnifedipine in the pipette solution (see alsoSection 2and[17]). As shown inFig. 4 and Table 1, 24–72 h exposureto ELFEFs had no significant effect on the biophysicalproperties of unitary currents through L or N channels.

3.5. Exposure to ELFEFs increases cell-membraneexpression of Ca2+channel subunit α1

The absence of any significant change in the unitary prop-erties of Ca2+-channels suggests that the enhanced Ca2+ in-flux in ELFEF-exposed cells is due to an increased numberof channels in the cell membrane. To test this hypothesis wemeasured the expression of the VGCC subunit�1 by West-ern blot analysis of plasma membrane-enriched microsomalfractions of GH3 and IMR32 cells.Fig. 3E shows that inEF-exposed cells the expression of�1 subunit was consis-tently increased over controls in both cell lines. Protein load-ing of Western blot was checked by evaluating the expres-sion levels of the membrane housekeeping protein aquaporinAQP-3 which resulted homogeneous in samples of controland ELFEF-exposed cells.

4. Discussion

Our data demonstrate that exposure to 50 Hz EFs en-hances cell proliferation in human neuroblastoma IMR32and rat pituitary GH3 cells. It also significantly inhibits pro-grammed cell death induced by puromycin or H2O2. Botheffects are abolished by global blockade of HVA Ca2+ chan-nels and markedly reduced by selective inhibition of L-typeCa2+ channels. The Ca2+-mediated effects of ELFEFs oncell proliferation and apoptosis are associated with markedincreases in Ca2+ current density and membrane expressionof the VGCC subunit�1 in both cell lines.

The EF effects on cell proliferation and apoptosis aredose-dependent at intensities ranging from 0.5 to 1.0 mT.


Fig. 4. Effects of ELFEFs on the biophysical properties of L-type and N-type Ca2+-channels in GH3 and IMR32 cells, respectively. (A) Representativemulti-channel recordings of L currents in control and ELFEF-exposed cells. (B) Slope conductance in ELFEF-stimulated channels (circles) is no differentfrom that of controls (squares). Values are reported inTable 1. C and D show corresponding data for N channels.

Lower intensities seemed to be ineffective, but the 0–1.0 mTrange was not systematically explored, so we cannot excludethe possibility of a window effect at low EF intensity.

The proliferative effects of ELFEF stimulation we ob-served in neuronal and neuroendocrine cells are consistentwith those reported for several cell lines[21–24]. In otherexperimental models, proliferation is instead unaffected[5,25–28] or even inhibited by EFs[29,30]. Moreover,EF stimulation per se does not influence the survival ofhippocampal neurons but, delivered in the presence of ni-tric oxide, it increases neuronal death[31]. These findingsclearly suggest that susceptibility to the proliferative effectsof EFs varies widely among cell types, and there is no sin-gle underlying mechanism that can be considered commonto all tissues. These differences should be considered in dis-cussion of the potential tumorigenic effects of EF exposureand data reported here on neural and neuroendocrine cellsoffer a contribution to the definition of this complex picture.

The inhibition of puromycin- and H2O2-induced apopto-sis produced by 50 Hzpulsating ELFEF stimulation in ourstudy is comparable to that described in U937 monocyticcells exposed to very high intensitystatic magnetic fields[5]. The latter type of exposure dose-dependently reducedetoposide-induced apoptosis with peak effects at an intensityas large as 6 mT. The significant inhibition of apoptosis ob-served in our study following 1 mT stimulation suggests that

EF generated by alternating currents might be more effectivethan static fields in regulating the programmed cell death.

Both the proliferative and anti-apoptotic effects of ELFEFstimulation appear to be related to changes in Ca2+ influxthrough VGCCs expressed on the cell membrane. Severalstudies have suggested that ELFEF exposure can alter the in-tracellular Ca2+ homeostasis. Significant increases in intra-cellular Ca2+ levels have been observed in various immune-cell models[10–12,32], but conflicting observations haveemerged from studies in neuroendocrine cells[13,14]. Therole of increased Ca2+ levels in EF modulation of cellproliferation and programmed cell death has also been sug-gested by previous reports[5,33]. In most cases, however,the EF-induced changes in intracellular Ca2+ concentra-tions were investigated with fluorescence imaging methods,which cannot clearly identify the source of Ca2+ increases,i.e. release from intracellular stores versus influx throughvoltage-dependent or independent cell-membrane ion chan-nels. Moreover, many of these studies were performed onnon-excitable cells in which changes in intracellular Ca2+concentration are produced by receptor-mediated mecha-nisms involving the inositol-1,4,5-triphosphate (IP3), andcell exposure to EFs was reported to significantly increaseIP3 levels[34]. The results we obtained with selective Ca2+channel blocking agents indicate that EF-induced modula-tion of apoptosis and cell proliferation is related to activity


of VGCCs. Previously reported patch-clamp data had failedto reveal any appreciable change in Ca2+ currents duringELFEF stimulation[35,36]. However, these observationswere limited to the study of effects induced by short-lasting(minutes) EF stimulation. In contrast, our data provide thefirst direct evidence that longer ELFEF exposure (24–72 h)induces a significant increase in transmembrane calciumcurrents that is due to enhanced expression of VGCCs.The absence of significant changes in the biophysical prop-erties of L and N channels exposed to ELFEFs excludesan effect on Ca2+ channel gating, and the enhanced Ca2+current density can therefore be attributed to an increase inthe number of the VGCCs. This conclusion is confirmedby Western blot data showing consistent ELFEF-inducedincreases in the expression of the VGCC subunit�1.

In summary, we have documented striking correlation be-tween the effects of ELFEFs on Ca2+ influx via VGCCs,proliferation and apoptosis in neural and neuroendocrinecells. The mechanisms through which the EF-induced en-hancement of Ca2+ influx can affect cell proliferation andprogrammed cell death were not specifically addressed inour study. However, data published by various groups showthat Ca2+ entry, particularly that flowing through L chan-nels, can activate signaling pathways leading to the expres-sion of genes that are essential for modulating cell differ-entiation, survival and apoptosis[5,37–40]. Future studiesshould therefore be aimed at identifying the specific intracel-lular pathways that transduce EF-induced changes in Ca2+influx into a signal capable of altering the fate (proliferationversus death) of exposed neurons and neuroendocrine cells.

Acknowledgements

The authors are grateful to Professor A. Musolino for su-pervision to the construction and the monitoring of the EFgenerating devices and to Mr. D. Mezzogori for skillful tech-nical assistance. This research was promoted and financedfrom I.S.P.E.S.L. (Istituto Superiore per la Prevenzione ela Sicurezza del Lavoro), MIUR (Ministero dell’Università,dell’Istruzione e della Ricerca) and UCSC.

References

[1] C. Polk, E. Postow, Handbook of Biological Effects of Electromag-netic Fields, CRC Press, New York, 1996.

[2] A. Lacy-Hulbert, J.C. Metcalfe, R. Hesketh, Biological responses toelectromagnetic fields, FASEB J. 12 (1998) 395–420.

[3] R.P. Liburdy, D.E. Callahan, J. Harland, E. Dunham, T.R. Sloma, P.Yaswen, Experimental evidence for 60 Hz magnetic fields operatingthrough the signal transduction cascade. Effects on calcium influxand c-MYC mRNA induction, FEBS Lett. 334 (1993) 301–308.

[4] J. Zhou, G. Yao, J. Zhang, Z. Chang, CREB DNA binding activationby a 50-Hz magnetic fields in HL60 cells is dependent on extra- andintracellular Ca2+ but not PKA, PKC, ERK, or p38 MAPK, BBRC296 (2002) 1013–1018.

[5] C. Fanelli, S. Coppola, R. Barone, et al., Magnetic fields increasecell survival by inhibiting apoptosis via modulation of Ca2+ influx,FASEB J. 13 (1999) 95–102.

[6] J. Walleczek, Electromagnetic field effects on cells of the immunesystem: the role of calcium signaling, FASEB J. 6 (1992) 3177–3185.

[7] R. Karabakhtsian, N. Broude, N. Shalts, S. Kochlatyi, R. Goodman,A.S. Henderson, Calcium is necessary in the cell response to EMfields, FEBS Lett. 349 (1994) 1–6.

[8] L.A. Coulton, A.T. Barker, Magnetic fields and intracellular cal-cium: effects on lymphocytes exposed to conditions for ‘cyclotronresonance’, Phys. Med. Biol. 38 (1993) 347–360.

[9] M.R. Cho, H.S. Thatte, M.T. Silvia, D.E. Golan, Transmembranecalcium influx induced by ac electric fields, FASEB J. 13 (1999)677–683.

[10] J. Walleczek, R.P. Liburdy, Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances45Ca2+ uptake in rat thymocytes: dependenceon mitogen activation, FEBS Lett. 271 (1990) 157–160.

[11] E. Lindström, P. Lindström, A. Berglund, K.H. Mild, E. Lundgren,Intracellular calcium oscillations induced in a T-cell line by a weak50 Hz magnetic field, J. Cell Physiol. 156 (1993) 395–398.

[12] E. Lindström, P. Lindström, A. Berglund, E. Lundgren, K.H. Mild,Intracellular calcium oscillations in a T-cell line after exposure toextremely-low-frequency magnetic fields with variable frequenciesand flux densities, Bioelectromagnetics 16 (1995) 41–47.

[13] E. Barbier, B. Dufy, B. Veyret, Stimulation of Ca2+ influx in ratpituitary cells under exposure to a 50 Hz magnetic field, Bioelectro-magnetics 17 (1996) 303–311.

[14] G.L. Craviso, J. Poss, C. Lanctot, S.S. Lundback, I. Chatterjee, N.G.Publicover, Intracellular calcium activity in isolated bovine adrenalchromaffin cells in the presence and absence of 60 Hz magneticfields, Bioelectromagnetics 23 (2002) 557–567.

[15] O.P. Hamill, A. Marty, E. Neher, B. Sakmann, F.J. Sigworth, Im-proved patch-clamp techniques for high-resolution current record-ing from cells and cell-free membrane patches, Pflügers Arch. 391(1981) 85–100.

[16] V. Carabelli, M. D’Ascenzo, E. Carbone, C. Grassi, Nitric oxide in-hibits neuroendocrine Ca(V)1 L-channel gating via cGMP-dependentprotein kinase in cell-attached patches of bovine chromaffin cells, J.Physiol. 5 (41) (2002) 351–366.

[17] M. D’Ascenzo, G. Martinotti, G.B. Azzena, C. Grassi, cGMP/proteinkinase G-dependent inhibition of N-type Ca2+ channels induced bynitric oxide in human neuroblastoma IMR32 cells, J. Neurosci. 22(2002) 7485–7492.

[18] J.H. Gerlach, D.R. Bell, C. Karakousis, et al., P-glycoprotein inhuman sarcoma: evidence for multidrug resistance, J. Clin. Oncol. 5(1987) 1452–1460.

[19] G. Glassmeier, M. Hauber, I. Wulfsen, F. Weisberg, C.K. Bauer,J.R. Schwarz, Ca2+ channels in clonal rat anterior pituitary cells(GH3/B6), Pflügers Arch. 442 (2001) 577–587.

[20] E. Carbone, E. Sher, F. Clementi, Ca currents in human neuroblas-toma IMR32 cells: kinetics, permeability and pharmacology, PflügersArch. 416 (1990) 170–179.

[21] J.D. Harland, R.P. Liburdy, Environmental magnetic fields inhibitthe antiproliferative action of tamoxifen and melatonin in a humanbreast cancer cell line, Bioelectromagnetics 18 (1997) 555–562.

[22] M. De Mattei, A. Caruso, G.C. Traina, F. Pezzetti, T. Baroni, V.Sallazzo, Correlation between pulsed electromagnetic fields exposuretime and cell proliferation increase in human osteosarcoma cell linesand human normal osteoblast cells in vitro, Bioelectromagnetics 20(1999) 177–182.

[23] M. Wei, M. Guizzetti, M. Yost, L.G. Costa, Exposure to 60-Hzmagnetic fields and proliferation of human astrocytoma cells in vitro,Toxicol. Appl. Pharmacol. 162 (2000) 166–176.

[24] V. Manni, A. Lisi, D. Pozzi, et al., Effects of extremely low frequency(50 Hz) magnetic field on morphological and biochemical propertiesof human keratinocytes, Bioelectromagnetics 23 (2002) 298–305.


[25] C.D. Cain, D.L. Thomas, W.R. Adey, 60 Hz magnetic field acts asco-promoter in focus formation of C3H/10T1/2 cells, Carcinogenesis14 (1993) 955–960.

[26] R. Higashikubo, V.O. Culbreth, D.R. Spitz, et al., Radiofrequencyelectromagnetic fields have no effect on the in vivo proliferation ofthe 9L brain tumor, Radiat. Res. 152 (1999) 665–671.

[27] L.I. Loberg, W.R. Engdahl, J.R. Gauger, D.L. McCormick, Cellviability and growth in a battery of human breast cancer cell linesexposed to 60 Hz magnetic fields, Radiat. Res. 153 (2000) 725–728.

[28] J.G. Robison, A.R. Pendleton, K.O. Monson, B.K. Murray, K.L.O’Neill, Decreased DNA repair rates and protection from heat in-duced apoptosis mediated by electromagnetic field exposure, Bio-electromagnetics 23 (2002) 106–112.

[29] S. Pacini, G.B. Vannelli, T. Barni, et al., Effect of 0.2 T staticmagnetic field on human neurons: remodeling and inhibition of signaltransduction without genome instability, Neurosci. Lett. 267 (1999)185–188.

[30] R.S. Stepanian, A.A. Barsegian, Zh.R. Alaverdian, G.G. Oganesian,L.S. Markosian, S.N. Airapetian, The effect of magnetic fields onthe growth and division of the lon mutant ofEscherichia coli K-12,Radiat. Biol. Radioecol. 40 (2000) 319–322.

[31] A. Boland, D. Delaoierre, D. Mossay, A. Dresse, V. Seutin, Effectof intermittent and continuous exposure to electromagnetic fields oncultured hippocampal cells, Bioelectromagnetics 23 (2002) 97–105.

[32] R.P. Liburdy, Calcium signaling in lymphocytes and ELF fields.Evidence for an electric field metric and a site of interaction involvingthe calcium ion channel, FEBS Lett. 301 (1992) 53–59.

[33] X. Bian, F.M. Hughes, Y. Huang, J.A. Cidlowski, J.W. Puteney Jr.,Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in inductionand suppression of apoptosis in S49 cells, Am. J. Physiol. 272 (1997)C1241–C1249.

[34] I.L. Korzh-Sleptsova, E. Lindström, K. Hansson Mild, A. Berglund,E. Lundgren, Low frequency MFs increased inositol 1,4,5-triphosphate levels in the Jurkat cell line, FEBS Lett. 359 (1995)151–154.

[35] P. Höjevik, J. Sandblom, S. Galt, Y. Hamnerius, Ca2+ ion transportthrough patch-clamped cells exposed to magnetic fields, Bioelectro-magnetics 16 (1995) 33–40.

[36] M. Obo, S. Konishi, Y. Otaka, S. Kitamura, Effect of magnetic fieldexposure on calcium channel currents using patch clamp technique,Bioelectromagnetics 23 (2002) 306–314.

[37] H. Bito, K. Deisseroth, R.W. Tsien, Ca2+-dependent regulation inneuronal gene expression, Curr. Opin. Neurobiol. 7 (1997) 419–429.

[38] P. Nicotera, S. Orrenius, The role of calcium in apoptosis, CellCalcium 23 (1998) 173–180.

[39] C. Morgado-Valle, L. Verdugo-Dıaz, D.E. Garcıa, C. Morales-Orozco, R. Drucker-Colın, The role of voltage-gated Ca2+ channelsin neurite growth of cultured chromaffin cells induced by extremelylow frequency (ELF) magnetic field stimulation, Cell Tissue Res.291 (1998) 217–230.

[40] R.E. Dolmetsch, U. Pajvani, K. Fife, J.M. Spotts, M.E. Greenberg,Signaling to the nucleus by an L-type calcium channel-calmodulincomplex through the MAP kinase pathway, Science 294 (2001) 333–339.

effects of 50 hz electromagnetic fields on voltage-gated ca2+ channels and their role in modulation...

Documents