Effects of electromagnetic fields on membrane ion transport of cultured cells Toshitaka Ikeharaa, Hisao Yamaguchia, and Hiroshi Miyamotob aDepartment of Physiology, The University of Tokushima School of Medicine, Tokushima, Japan; and bDepartment of Life, Environment and Information, Faculty of Domestic Science, Tokushima Bunri University, Tokushima, Japan Abstract:We have studied the mechanisms of ion transport mediated by Na+/K+-pump and Na+,K+,Cl--cotransport pathway of HeLa cells using Rb+ as an analog for K+, and proposed models of binding of ions for the transport pathways. Also, we clarified the relation between ion and water movements in the cells. Based on these findings, we have studied the effects of homogeneous and time-varying magnetic fields on the ion transport activity. The research presented here covers (i) brief explanations of our kinetic studies on the ion transport pathways for promoting understanding of the effects of magnetic fields on the pathways, (ii) our and other reports of the effects of magnetic fields on ion transport systems. J. Med. Invest. 45:47-56 1998 Keywords:magnetic field, Na+,K+ pump, K+ channel, Ca2+ movement, Na+,K+,Cl--cotransport, volume regulation INTRODUCTION Cellular K+ transport takes place mediated by pathways in the cell membrane. In the present paper, we reviewed mechanisms of the major pathways of K+ transport in the cell membrane, and the biological effects of exposure to magnetic or electromagnetic fields on the functions of these pathways. The pathways consist of Na+,K+ pump, Na+,K+, Cl--cotransport pathway and K+ channels as well as other ion channels. In many cultured cells, K+ influx is chiefly mediated by the Na+,K+ pump (ouabain-sensitive), and so the pump activity strongly influences the intracellular concentrations of mono-valent ions and membrane potential. The presence of Na+,K+,Cl--cotransport pathway has been reported in a variety of cells, and the cotransport system is known to be specifically inhibited by certain diuretics such as bumetanide and furosemide. These agents are known to suppress Na+ reabsorption at the ascending limb of Henle's loop of the kidney. Ca2+dependent K+ channels are present in various types of excitable and non-excitable cells. Activity of the K+ channels contributes to electrical characteristics and many functions of these cells. K+ flux through the cotransport pathway and K+ channel is known to be activated when many types of cells are incubated in hypotonic or high K+ medium. There have been many reports on the biological effects of static or DC magnetic fields and time-varying magnetic or electromagnetic fields on membrane cation transport and related functions. These studies indicate that the effects of exposures to various magnetic fields are not identical. While some reports show an inhibitory effect by the fields, others show activation, and still others no significant influences on the cation transport. In recent years, many in-vestigators have tested the effect of exposure to a magnetic field on Ca2+ influx across the cell mem-brane or intracellular Ca2+ movements. The research reviewed here covers studies on the kinetics of membrane ion transport and the magnetic field exposure-induced effects on ion transport. We also discuss interactions of magnetic field-exposure with signal transduction events at the cell surface and in cytosol. METHODS Electromagnet The magnetic fields we used were produced by an electromagnet designed and set up by Hitachi Metal Indust. Co. (Tokyo, Japan). The apparatus consisted of a couple of vertically arranged coils attached with pole pieces of round polar faces with a large and a small diameter of 10 and 5cm, respec-tively (Fig.1). In proportion to coil current, static magnetic fields of flux densities of 0.3 to 1.7 T were produced in the gap between the large polar faces. Similarly, a magnetic field of about 2T at maximum was produced with the small polar faces. Time-varying magnetic fields were produced by automatic switching of the coil current with an elec-tronic device, which enabled us to change the magnetic flux density from 0.07 to 1.7 or 2T in about 1s (using the large or small polar faces). Time intervals of the changes were chosen to be 3 s or longer, since it took more than2s for eddy current to return to zero after switching off. The density of eddy current induced by switching on was 32 and 57mA/m2, the maximum value;and that for switching off was -13and -17mA/m2 respectively, the minimum value, when the large polar faces were used (1). Microfluorometry. Changes in the electrical charge on the cell surface that occurred when cells were exposed to the time-varying magnetic fields were monitored with a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin, by a slightly modified method of Pal et al. (2). Membrane potential of cells was assayed with a fluorescent agent, 3,3'-dipropyl-thiadicarbocyanine iodide (diS-C3-(5)), by a modified method of Wright et al.(3). The intracellular Ca2+concentration was determined and analyzed with a fluorescent probe, 1-(2-(5'-carboxyl-oxazol-2'-yl)-6-aminobenzofuran-5-oxy)-2-(2'-amino-5'-methyl-phenoxy)ethene-N,N,N',N'-tetraacetic acid, penta-acetoxymethyl ester (Fura2-AM) by the method of Grynkiewicz et al. (4). K+ transport measurement First, the usefulness of Rb+ as an analog of K+ in membrane transport was tested. Extracellular K+ (K+e) was totally or partly replaced with Rb+, and movement of the cation across the plasma membrane was investigated in HeLa cells. Cells were proven to transport Rb+ to the same extent equally as they did K+ (5), and Rb+ and K+ uptakes were almost equally sensitive to various concentrations of ouabain (5) or furosemide (6). These cations were shown to bind to the external K+ site on the ouabain-sensitive pump with the same affinities (7), and a similar result was obtained for the furosemide-sensitive pathway(6). Based on the results, we used non-radioactive Rb+ as a tool to investigate membrane ion transport in HeLa cells. Fig.2 is a schematic presentation of the major pathways of K+ influx through the mem-brane of HeLa cells. About 50-70% of the total Rb+ uptake was inhibited by ouabain and about 70-80%of ouabain-insensitive Rb+ uptake was inhibited by furosemide, indicating that about 50-70% of the Rb+ uptake was mediated by the Na+-pump, and about20-40% by the cotransport pathways. The fraction of the total K+ influx mediated by K+ channels was not determined, since K+ permeability could be measured exactly only by the patch-clamp technique. MECHANISMS OF ION TRANSPORT Na+,K+ pump Kinetic parameters and mechanism of the active cation transport in HeLa cells have been studied and the presence of two external K+ sites per Na+/K+-pump demonstrated. The affinities of the K+ sites for Rb+ as a congener of K+ are almost the same with that of K+. Na+e inhibits ouabain-sensitive Rb+ influx competitively, whereas intracellular K+ (K+c) is not inhibitory. Na+ and K+ are exchanged alter-nately through the pump by a binary mechanism (7). We have proposed a model system transporting the two solutes in consecutive sequence. Similarly, Sachs (8) has concluded that the kinetic behavior of the Na+ pump considered as a bisubstrate reaction with Na+ and K+ as substrates can be adequately described by a consecutive model with the modification that the Na+ binding sites can return from outside to inside even though no Na+ is binding. The active Rb+ influx can also be supported by oxidative metabolism and is regulated as a function of the cellular ATP content up to the control level, and2Rb+ ions move concomitantly at the expense of one ATP molecule (9). Na+,K+,Cl--Cotransport The Na+ gradients generates the driving force for symports and antiports of many essential substances. One of the Na+-dependent symports is electrically silent Na+,K+,Cl- cotransport known to be important for Na+ reabsorption in Henle's loops of the renal tubules. This cotransport pathway is specially sensi-tive to loop diuretics such as furosemide, bumetanide, and piretanide. Furosemide slightly but definitely inhibited Rb+ uptake in HeLa cells, but K+ efflux was not influenced (5);showing asymmetry of the ion movements on cis and trans sides of the mem-brane, different from results reported for mouse3T3 cells (10). Transmembrane gradients of the chemical potentials of Na+ could be the primary driving force of the furosemide-sensitive Rb+ influx (JRb) (11). We have examined JRb into HeLa cells as functions of extracellular Rb+, Na+ and Cl- concentrations. Rate equations and kinetic parameters, including the apparent maximum JRb, the apparent values of Km for the three ions and the apparent Ki for K+, were derived. We proposed a general and a specific furosemide-sensitive cotransport model. According to the model, one Na+ and one Cl- bind concurrently to their sites followed by one K+(Rb+) and another Cl-. After completion of the ion bindings, Na+, K+(Rb+) and Cl- show synchronous transmembrane movement in a ratio of 1:1:2. One unit molecule of this furosemide-sensitive cotransport system has one Na+, one K+ and two Cl- sites with different affinities (6). In different types of cells, Na+, K+ and Cl- are also transported across the cell membrane at a stoichiometry of 1:1:2 (12-14), and some kinetic parameters for the diuretic-sensitive cotransport in relation to extracellular ion have been estimated(15, 16). The cotransport of HeLa cells has also been studied by measuring JRb as a function of the cellular ATP and the external Rb+ and Na+ concentrations. ATP stimulates the cotransport by increasing the affinity of the cotransport pathways, and two ATP molecules are needed for uptake of one Rb+ by the cotransport pathway. Again, we have proposed a general and a specific model with respect to cellular ATP, and stated that cyclic AMP has no effect on the cotransport in HeLa cells (17). The effects of K+c and Na+c on the cotransport pathways were also studied. Extracellular K+ stimu-lates and Na+ inhibits JRb, without any change in the affinities of the pathway for Rb+e and Na+e (18). Rb+ uptake occurs when Rb+ binds to the external K+ site of the pathway after binding of internal K+ to its regulatory site. In contrast to HeLa cells, an increase in Na+c or decrease in K+c stimulates JK (furosemide-sensitive K+ influx) in the absence of exter-nal Na+ in human erythrocytes (15). Furosemide-sensitive 86Rb+ influx into human erythrocytes in vivo is negatively correlated with the concentration of external Na+e(19). Cell Volume Regulation in Relation to K+ Channel Addition of nystatin (non-voltage dependent channel forming ionophore) causes reversible changes in the cellular Na+ and K+ contents and cell volume of HeLa cells (20). These changes are inhibited by sucrose, and Donnan and osmotic equilibria are attained. The K+ content, the cellular free-Ca2+ con-centration ([Ca2+]i) and the cell volume of HeLa cells increase after a latent period following change to an isosmotic Na+-free, high K+ medium (21). When [Ca2+]i was extremely reduced by preloading BAPTA-AM, the K+ uptake decreased to the level reduced in the presence of quinine. The quinine-sensitive pathway depends on cellular Ca2+ and its major component is Cl--dependent, whereas the quinine-insensitive pathway is independent of the Ca2+ and unselective for anions (22). When medium osmolality is decreased, suspended HeLa cells instantly swell and then K+ loss and regulatory volume decrease (RVD) occurs, while attached HeLa cells instantly swell and then rapidly lose water and K+, before slowly gaining them again (23). Hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca2+-dependent K+ channel. The initial increase in K+c is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca2+-dependent K+ channels. We have demonstrated by patch clamp technique that the K+ channel of HeLa cells has characteristics of strong inward rectification, small conductance, and dependence on intracellular Ca2+ (24, 25). The inward K+ currents through the channel at fixed membrane potentials are highest in early G1 and then decrease with time to a minimum in the S phase, increasing again in the M phase. The capacitance and the permeability change with progression of the cell cycle. We did not show the presence of other types of K+ channels in HeLa cells. EFFECTS OF MAGNETIC FIELDS ON ION TRANSPORT Effects of magnetic fields on Na+ pump. Based on the characteristics of the membrane of HeLa cells stated above, we tested the effects of dif-ferent magnetic fields on cation transport of the cells. However, no significant effects on ouabain-sensitive (active) and-insensitive (passive) Rb+ influx into HeLa cells on exposure to homogeneous static magnetic fields of flux density 0 to 1.6 T were detected at 37°C (26). There were no significant effects on either Rb+ influxes of the static magnetic field of 1.5 and 2T at different temperatures from 15 to 42°C, and soevidence for the membrane phase transition was not found in this temperature range (26, 27). The Arrhenius plot of the data showed no significant influence of the exposures to the activation energy (28). The mean activity of Na+, K+ ATPase and Ca2+ATPase of rat diaphragm muscle homogenates is reported to be significantly higher in the static mag-netic field (0.02T)-exposed group, but that of Mg2+ATPase is not significantly changed (29). Next, we tested the effect on the ouabain-sensitive Rb+ influx of exposure to the strong time-varying magnetic fields of 1.7T maximum. When durations of the periods of"switching-on"and"switching-off" of coil current were limited to1min or longer, there was no significant effect of the magnetic field (1).However, when these times were shortened to 3 sec, the Rb+ influx was suppressed significantly. The inhibition of the active Rb+ influx increased with time during exposure for 2hr. It is likely that the inhibition of Na pump by exposure to the time-varying magnetic field has a close relation to change in the surface properties of the cell membrane. With respect to the effect of time-varying magnetic fields on active cation transport, ouabain-sensitive Na+ fluxes across stripped rabbit colon epithelium have not significantly been influenced by pulsed magnetic fields (30). Na+,K+,Cl--Cotransport We could not show any significant effect of the static magnetic field at less than 2T on ouabain-insensitive Rb+ influx, suggesting no significant effect of the magnetic field on Na+,K+,Cl--cotransport (1). No significant influence of pulsed magnetic field on the furosemide-sensitive 86Rb+ influx into human eryth-rocytes has been reported (30). Effects of magnetic fields on K+ channel and Ca2+ influx. K+ channel activity plays an important role in producing the resting membrane potentials. We tested the effects of the strong time-varying magnetic field on K+ uptake of HeLa cells incubated in an isosmotic high K+ medium. The uptake was demonstrated to take place mainly mediated by Ca2+-dependent K+ channel, since specific inhibitors of the channel such as quinine, charybodotoxin and iberiotoxin sig-nificantly suppressed the uptake (31). It is known that cells are depolarized in the high K+ medium, and the intracellular Ca2+ concentration ([Ca2+]c) is increased. However, the exposure to the magnetic field significantly reduced cellular K+ uptake and completely suppressed the increase in cell Ca2+ in the high K+-medium. The inhibition of K+ uptake was not restored by addition of Ca2+ ionophore ionomycin. These results suggest that exposure-induced inhi-bition of the K+ channel activity was related to inhi-bition of intracellular Ca2+ mobilization. Furthermore, the results suggest that cell swelling in the high K+ medium can be significantly suppressed by the ex-posure. This is the first study to report the effects of magnetic fields on K+ channel activity. Effects of Magnetic Fields on Molecular Events during Receptor-Mediated Signal Transduction including Ca2+ Uptake. Ca2+ signaling is well documented in many types of cells as a part of the second messenger system. Recently, the number of reports on the effects of magnetic fields on Ca2+ signal transduction has increased. For example, ELF magnetic field expo-sures have no effect on concanavalin A-stimulated calcium uptake in rat thymocytes (32). There is no biologically significant effect of weak combined dc-ac magnetic fields on the binding of calcium to calmodulin and MLCK (myosin light chain kinase) activity (33). But, other reports show the influences of exposures to various magnetic fields. During exposure to an ELF field (50Hz, 0.1 or 0.15mT) an acute response is observed with oscillatory increases in intracellular Ca2+ and inositol 1,4,5-triphosphate (IP3) levels in T cells or Jarkat cell line, which subsides when the magnetic field is turned off (34 -36). Similarly, 45Ca2+ uptake by lymphocytes increases during expo-sure to another ELF field (16 or 60Hz) (37, 38). Low amplitude combined magnetic field is capable of increasing net Ca2+ flux in human osteoblast-like cells and the increase is frequency-dependent (39). In lymphocytes, an application of a low frequency electric field exerts little influence on the early release of Ca2+ from intracellular stores in Ca2+-free buffer, but enhances Ca2+ influx through the plasma mem-brane in the presence of external Ca2+ (40). The magnetic field affects membrane depolarization, increases action potential discharge and reduces uptake of Ca2+ into ganglia and isolated neurons of Helix pomatia (41). Effects of Magnetic Fields on Membrane Electrical Properties. Membrane electrical properties such as membrane surface charge, membrane potential and so on may be directly influenced by eddy current induced by changing the flux density of magnetic field. There have been studies testing the effects of static or dc magnetic fields on some of the electric properties. Rat diaphragm has shown less negative resting po-tential, and the amplitude and overshoot of action potential is significantly decreased upon exposure to a static magnetic field (29). Exposure to another static magnetic field of magnetic gradient of about1mT/mm blocks more than 70% of the action po-tential of sensory neurons in culture (42). When a mammalian cell line U937 is exposed to a pulsed magnetic field of peak flux density of 0.63mT, cells exhibit increased negative surface charge, whereas membrane hydrophobicity is not significantly altered (43). In our study, microfluorometry of HeLa cells loaded with a fluorescent pH indicator 4-heptadecyl-7-hydroxycoumarin (Fig.3) and a membrane potential indicator diS-C3-(5) (Fig.4) revealed increases in the negative surface charge during exposure to the strong time-varying magnetic fields. Simultaneously observed change in the K+(Rb+) fluxes would close-ly relate to the change in the membrane electric properties (1). In contrast, myogenic cells exposed to a static magnetic field show no changes in mem-brane electrical parameters such as conductivity and permittivity (44). Other Results related to the Effects of Magnetic Fields. Static magnetic fields have been reported to affect the diffusion of biological particles in solutions by inducing Lorentz force or Maxwell stress. Lorentz force would influence the diffusion of charged particles such as various ions including plasma proteins (45). In fact, it has been reported that changes in electrical conductivity of CaCl2 solution are caused by exposure to static magnetic fields (2.3-350mT) (41). Recently, we showed that ELF magnetic field (80mT and50Hz) affected the molecular structure of membrane protein in HeLa cells, as determined by Fourier transform infrared spectroscopy (46).The exposure-induced modification of membrane protein structure may be related to the change in membrane electrical properties. Santoro et al. have reported a decrease in membrane fluidity and re-organized cytoskeletal components on exposure to ELF magnetic field in human B lymphoid cells (Raji) (47). Therefore, ELF magnetic fields would influence the structure of protein molecules composing the biomembrane. Positive and negative data have been published on the effects of various magnetic fields on ion transports or membrane properties related to the transports. Liburdy has shown that the effects of ELF magnetic fields are due to induced electric fields rather than flux density (40). We basically agree with this. So, electric field or current induced by time-varying magnetic field could affect more strongly ion trans-ports in the magnetic field. Strong eddy current in the medium induced by changes in the flux density could affect electric properties of the cell surface, but the current would not penetrate into the cells since the cell bilayer act as an electrical insulator. A low frequency magnetic field in combination with a static magnetic field increases the number of receptors of the type II insulin-like growth factor (IGF-II) on the membrane of osteosarcoma cells(48). This result supports our hypothesis. On the other hand, according to Faraday's law, the current induced by the magnetic fields inside the cells is far weaker than the one induced in the extracellular space. But, the possibility of a direct effect of eddy current in the cells cannot be totally excluded. Because, we found that the increase in [Ca2+]i is strongly inhibited in cells incubated in a Ca2+-free medium by the time-varying magnetic field. Liburdy (49) has detected an increase in cal-cium uptake into mitogen-stimulated rat thymocytes (mature) and human lymphocytes during exposure to 60 Hz ELF field or high-field NMR (Nuclear Magnetic Resonance). As NMR fields contain a time-varying magnetic field, this result implies that the time-varying field of NMR is an operative com-ponent responsible for the effect on calcium transport. Next, we concerned ourselves with a steady com-ponent of magnetic fields of NMR and MRI (NMR imaging system) clinically applied. The maximum flux density of the steady component of high-field MRI is about2T, similar to that produced by our electromagnet. The static magnetic field was shown not to affect either ouabain-sensitive or-insensitive K+ uptake in HeLa cells. Many investigators have also shown insignificant influences on various cellular functions of exposure to a similar magnitude static or DC magnetic field. However, MRI magnetic fields contain a non-homogeneous (gradient) component in addition to homogeneous static and time-varying components. Further investigation is needed on the effects on cellular functions of the gradient field that exerts Maxwell stress. CONCLUSION Figure5 depicts the molecular events during receptor-mediated signal transduction in cultured cells, and also indicates the possible places that could be affected when these cells are exposed to magnetic fields. We propose these sites based on our data and those of others and describe them in detail in the legend to Fig.5. Incubation in an isotonic high K+ medium depolarized the membrane of HeLa cells, and the depolarization would increase the intracellular Ca2+ concentration [Ca2+]i by Ca2+ influx through Ca2+ channels of the membrane or Ca2+ release from intracellular Ca2+stores. Exposure to the time-varying magnetic field significantly suppressed the increase in [Ca2+]i and partly inhibited the K+ influx through Ca2+-dependent K+ channels, suggesting inhibition of Ca2+ influx and/or Ca2+ release by the exposure. However, the inhibition of K+ influx would be due to a direct effect of the exposure on K+ channels rather than sup-pression of the increase in [Ca2+]i (31). Liburdy et al. (40) have reported that ELF magnetic field affects Ca2+ uptake across the cell membrane, but Ca2+ release from the Ca2+ stores is not influenced. This is because electric field or eddy current induced by the magnetic field does not effectively penetrate the outer cell membrane. 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FEBS Lett 377:31-36, 1995 52. Liu PS, Lin YJ, Kao LS:Caffeine-sensitive calcium stores in bovine adrenal chromaffin cells. J Neurochem56:172-177, 1991 Received for publication July 17, 1998 ; accepted July 31, 1998. 1 Address correspondence and reprint requests to Hisao Yamaguchi, Ph.D., Department of Physiology, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-886-33-9443.