Cellular membrane potentials induced by alternating fields

Membrane potentials induced by external alternating fields are usually derived assuming that the membrane is insulating, that the cell has no surface conductance, and that the potentials are everywhere solutions of the Laplace equation. This traditional approach is reexamined taking into account membrane conductance, surface admittance, and space charge effects. We find that whenever the conductivity of the medium outside the cell is low, large corrections are needed. Thus, in most of the cases where cells are manipulated by external fields (pore formation, cell fusion, cell rotation, dielectrophoresis) the field applied to the cell membrane is significantly reduced, sometimes practically abolished. This could have a strong bearing on present theories of pore formation, and of the influence of weak electric fields on membranes.     

Orientation behavior of retinal photoreceptors in alternating electric fields

In alternating electric (AC) fields, particles experience polarizing effects that induce dipoles that orient elongated specimens either parallel or perpendicular to the field lines. In this work we studied the behavior of photoreceptor cells' rod outer segments (ROS) in AC fields of different frequencies. We showed that at low frequencies, ROS orient parallel to the field, whereas at higher frequencies they orient perpendicular to the field lines (in the frequency range from 100 Hz to 10 MHz). We found this behavior to be dependent on the physiological state of cells (due to modifications in their electrical properties). To simulate cell damage, the membrane conductivity was changed by treating the cell with gramicidin A, which resulted in a decrease of cytosol conductivity and, consequently, in a change of the orientation behavior of the treated cells. The change of cell orientation with cytosol conductivity is rather sharp, suggesting the potential of the method for accurate evaluation of the cell physiological status. We modeled the interaction between ROS and AC fields approximating the rod cell by a prolate spheroid with a very long axis. The internal compartment of the ellipsoid was considered to be filled with an inhomogeneous medium consisting of alternating layers of membrane and cytoplasm as media modeling the disks. This theoretical model proved to be in good agreement with the experimental results and enabled the derivation (by fitting with the experimental results) of the membrane and cytosol parameters for normal and damaged cells.     

Na,K-ATPase function in alternating electric fields.

Alternating currents affect ion transport processes and ATP splitting through changes in the activation of the membrane Na,K-ATPase. Both processes vary with the frequency, and the effective range includes the environmental 60 Hz. ATP splitting by Na,K-ATPase suspensions decreases for the enzyme under normal conditions, with the maximum effect at 100 Hz. ATP splitting increases when the enzyme activity is lowered to less than half its optimal value by changes in temperature, ouabain concentration, etc. These observations can be explained by the effects of the ionic currents on ion binding at the enzyme activation sites. Such a mechanism could account for the effects of electromagnetic fields on cells, as the transmembrane enzyme can convey the effect of an extracellular signal into the cell via ionic fluxes, and the measured threshold field is within the range of reported biological effects.     

Schwan equation and transmembrane potential induced by alternating electric field.

The transmembrane potential generated by an alternating electric field (ac) depends strongly on the frequency of the field and can be calculated using the Schwan Equation. We have measured the critical electric breakdown potential, delta psi crit, of the plasma membrane of murine myeloma cell line (Tib9) using ac fields, by monitoring the entry of a fluorescence probe, propidium iodide, into the cells. This dye is weakly fluorescent in solution but becomes strongly fluorescent when it binds to DNA. Experiments were done under a microscope by direct visual examination of single cells or by examining photographic prints. When an ac field reached the intensity, Ecrit, that generated a maximal membrane potential delta psi max, equal to or greater than the delta psi crit, the membrane was perforated at the two loci facing the electrodes. The dye diffused into the cell, giving rise to two bright, narrow bands, which expanded to the whole cell in 1-3 min. delta psi crit's were measured in three media of different resistivities, rho ext, (52,600, 7,050, and 2,380 omega cm), over the range of 0.1-300 kHz, with the field duration of 200 ms. Regression analysis based on the Schwan Equation showed that in a medium of given resistivity, the delta psi crit was constant over the frequency range studied. When the capacitance of the membrane, Cmembr, was taken to be 0.90 microF cm-2, the resistivity of the cytoplasmic medium, rho int, was determined to be 910-1,100 omega cm. The delta psi crit were 0.33, 0.48, and 0.53 V, respectively, for the three media in decreasing resistivities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Miniature-probe measurements of electric fields induced by 60 Hz magnetic fields in rats

Extremely-low-frequency (ELF) magnetic fields interact with an animal by inducing internal electric fields, which represent the internal dose from an external exposure. In this study, an electric field probe of approximately 2 mm resolution was used to measure fields induced in rat carcasses by a 60 Hz magnetic field at 1 mT. With the rat lying on its side, the probe was inserted through a small hole in the body wall, and scanned at 5 mm increments from the side with frontal and axial exposure (field horizontal) and from the front with lateral exposure (field vertical). The induced electric field declined from a maximum at the entrance to the abdomen and crossed zero to negative (180° phase shift) values within the body as expected. In general, the magnitudes of the measurements inside the abdomen were less than expected from whole-body calculations that used homogeneous-ellipsoidal models of a rat in the three orientations. The low measurements did not appear to be explained by perpendicular field components, by conductivity differences between the tissue and the probe path, or by air in the lungs. The low measurements probably result from inhomogeneities in actual rats that include conductivity differences between tissues and biological membranes. For example, an alternative model considered the abdominal cavity to be electrically isolated from the body by the diaphragm and the peritoneum and calculations from this model were in better agreement with the measurements inside the abdomen (than were the whole-body calculations). Therefore, inhomogeneities in conductivity and biomembranes such as the peritoneum should be considered in order to fully understand ELF-induced field dosimetry. © 1996 Wiley-Liss, Inc.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0. We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Frequency specific modulation of bone adaptation by induced electric fields

A frequency specificity for the response of bone tissue to a physical stimulus is proposed. This is obtained by comparing the spectral power of exogenously induced electric fields to the efficacy of those fields to inhibit immobilization induced bone loss in an in vivo model of skeletal adaptation. Analysis of a family of related waveforms shows that the effectiveness of the induced electric fields could be related to the induced spectral power below approximately 75 Hz. The analysis suggests that bone tissue may be extremely sensitive to induced power levels at or below this frequency, as amplitude variations of less than a factor of two within this range correspond to significantly different bone remodeling responses. The analysis also suggests that bone tissue may be strongly frequency selective, with bone capable of responding specifically to induced power in this osteogenic frequency band, even though the band includes less than 0.1% of the total induced power. As normal functional activity generates strain components encompassing this osteogenic frequency band, a distinct frequency selectivity may indicate that the tissue response is tuned to a specific endogenous stimulus. A detailed characterization of the frequency response of bone tissue could well point to the primary source for the control of the cells responsible for functional adaptation in the skeleton.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0 We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Threshold effects observed in conformation changes induced by electric fields

Single-stranded polynucleotides are used as model systems for the investigation of conformational changes induced by electric fields. It is demonstrated that the single-strand helix–coil transition in poly(A), poly(dA), and poly(C) can be induced by application of high electric fields. The transition is measured by UV absorbance using polarized light at an angle of 54.8° with respect to the vector of the electric field and by electrodichroism. A linear increase of the absorbance, reflecting the helix-to-coil transition, is observed at increasing field strength. When ions are added to the polymer, electric fields do not induce conformation changes, unless a threshold value of the electric field strength E₀ is exceeded. At field strengths above this threshold, the degree of transition is a linear function of the increase in field strength. The threshold values E₀ show a linear increase with the logarithm of the ion concentration. Bivalent ions cause thresholds at much lower ion concentrations than mo-novalent ions. The shielding efficiency of ions is correlated to the binding affinity of these ions to the polymer. The conformation changes induced by the field and the existence of thresholds can be explained on the basis of dissociation field effects. Similar threshold effects may be expected for other macromolecules as well as for membrane structures and may be important in the regulation of bioelectricity.     

Permeabilization of tumor cells induced by pulsed electric fields in vitro

The permeabilization of tumor cells in vitro under the action of pulsed electric fields with a duration of 6 mks in the range of amplitudes 1-7 kV/cm was studied. In the mode of excitation in the ambience of localized plasma discharge in a chamber of special design, an enhanced damage to cells in suspension was observed. It is assumed that the enhancement is due to the synchronous action of the electric field and acoustic shock wave pulses. In the mode without the plasma breakdown of ambience, when the pulse duration of electric field of intensity of 1-2 kV/cm was increased to 60 mks, the efficiency of permeabilization increases nearly by one order. The experimental results are compared with the known theoretical models of cell membrane electroporation.     

Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields.

Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane potential perturbations induced in tissue cells by pulsed electric fields

Pulsed electric fields directly influence the electrophysiology of tissue cells by transiently perturbing their transmembrane potential. To determine the magnitude and time course of this interaction, electrotonic cable theory was used to calculate the membrane potential perturbations induced in tissue cells by a spatially uniform, pulsed electric field. Analytic solutions were obtained that predict shifts in membrane potential along the length of cells as a function of time in response to an electrical pulse. For elongated tissue cells, or groups of tissue cells that are coupled electrotonically by gap junctions, significant hyperpolarizations and depolarizations can result from millisecond applications of electric fields with strengths on the order of 10–100 mV/cm. The results illustrate the importance of considering cellular cable parameters in assessing the effects of transient electric fields on biological systems, as well as in predicting the efficacy of pulsed electric fields in medical treatments. © 1995 Wiley-Liss, Inc.     

Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.     

The magnetic and electric fields induced by superparamagnetic magnetite in honeybees

Hairs on the abdomen of honeybees contain dendrites and a rod and ring structure composed of black particles, presumed to be superparamagnetic (SPM) magnetite. The rod and ring were divided into compartments and each compartment approximated by a dipole. The magnetic fields were calculated at a point P at various locations for a change of the external geomagnetic field from zero to 0.5 G in 0.1 s. The magnetite amplifies the external field at the rod/ring-dendrite interface. The induced electric field and potential difference for a small circular area are in the order of 10^–7 V/m and 10^–13 V respectively. Mechanisms are proposed for amplifying the electric fields in the dendrite and in an integrating nerve fibre. A hypothesis is developed for associative learning of visual and magnetic stimuli. If magnetic and visual inputs are associated in the ganglion and in the brain, very small changes of either magnetic or visual inputs could be perceived. A bee could sequentially follow the images associated with magnetic gradients on a cloudy day and find the food source.This paper is dedicated to the memory of the late Prof. Dr. W. Reichardt, an outstanding scientist and personality, who will be greatly missed by all who knew him and his work     

Sizing of vesicles by electric birefringence

A study of the transient, electrically induced birefringence of aqueous suspensions of phospholipid vesicles (liposomes) has indicated a rapid and sensitive method of measuring the size and electrical polarizability anisotropy of these entities.     

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles: Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.