Galvanotropic and galvanotaxic responses of corneal endothelial cells.

The effects of weak electric fields (E-fields) on cultured rabbit corneal endothelial cells were studied. The cells responded to steady E-fields (2-6 V/cm) by elongating their somata 90 degrees to the field (galvanotropism) and by migrating (galvanotaxis) towards the anode. During these directional movements, pseudopodia and ruffled membranes formed preferentially on the anodal side of the cells, while they retracted on the cathodal side. Fluorescent labelling for actin showed many stress fibers aligned parallel to the long axes of the elongated cells and few aligned toward the anodal direction. Fluorescent labelling for vinculin showed the abundance of cell-to-substratum adhesion foci at the termini of the stress fibers. Galvanotropic and galvanotaxic cellular movements were inhibited by cytochalasin D (0.1-0.5 microgram/mL) and the calmodulin antagonist, W-7 (80 mumol/L). These results suggest that E-field induced directional movements of corneal endothelial cells constitute a calmodulin-dependent, active (not passive) process.

Diatom response to extremely low-frequency magnetic fields.

Reports that extremely low-frequency magnetic fields can interfere with normal biological cell function continue to stimulate experimental activity as well as investigations into the possible mechanism of the interaction. The "cyclotron resonance" model of Liboff has been tested by Smith et al. (Bioelectromagnetics 8, 215-227, 1987) using as the biological test system the diatom Amphora coffeiformis. They report enhanced motility of the diatom in response to a low-frequency electromagnetic field tuned to the cyclotron resonance condition for calcium ions. We report here an attempt to reproduce their results. Following their protocol diatoms were seeded onto agar plates containing varying amounts of calcium and exposed to colinear DC and AC magnetic fields tuned to the cyclotron resonant condition for frequencies of 16, 30, and 60 Hz. The fractional motility was compared with that of control plates seeded at the same time from the same culture. We find no evidence of a cyclotron resonance effect.

Effects of steady electric fields on human retinal pigment epithelial cell orientation and migration in culture.

Low-level, steady electric fields of 6-10 volts/cm stimulated directional orientation and translocation of cultured human retinal pigment epithelial cells. The orientative movements (galvanotropism) consisted of somatic elongation of the cells into spindle shapes, followed by pivotal alignment orthogonal to the field. The anodal edges of the cells underwent retraction of their plasmalemmal extensions, while the cathode edges and the longitudinal ends developed lamellipodia and ruffled membranes. These tropic movements were followed by a translocational movement (galvanotaxis) of the cells towards the cathode. Staining of these migrating cells for actin showed the accumulation of stress fibers at the leading (cathodal) edge, as well as at the longitudinal ends of the elongated somata. These results suggest that endogenous, biologically-generated electric fields (eg., injury currents) may play a role in the guidance and migration of retinal pigment epithelial cells after retinal injury.

Search for ion-cyclotron resonance in an Na(+)-transport system.

Colonic tissue from the turtle (Pseudemys scripta) was exposed in a Ussing chamber to simultaneously applied static and time-varying magnetic fields. Transepithelial differences of potential were monitored as frequency of the AC field was varied continuously or in discrete steps from 3 to 770 Hz. Density of the DC field was varied from 10 to 220 microT and that of the AC field from 1 to 20 microT. Short-circuit currents through tissue were monitored for changes that might have been observed under ion-cyclotron resonance (ICR) conditions for each of several ions: H+, Li+, Na+, K+, Ca2+, and Cl-. No discernible changes of transepithelial current were observed. The negative findings are discussed in relation to positive and negative findings that have appeared in the literature.

Movements of cultured corneal epithelial cells and stromal fibroblasts in electric fields.

The effects of an externally applied direct-current electric field on the movement of cultured rabbit corneal epithelial cells and stromal fibroblasts were studied. After a latency of approximately 20 minutes in an electric field, both epithelial cells and stromal fibroblasts became spindle shaped and underwent galvanotropism by aligning their long axes perpendicular to the applied electric field. The electric field stimulus thresholds for galvanotropic movements in epithelial cells and stromal fibroblasts were 4V/cm and 6 V/cm, respectively. After an additional latency of 30 minutes, both cell types manifested galvanotaxic movements: epithelial cells commenced migration in the cathodal (downfield) direction and stromal fibroblasts in the anodal (upfield) direction. For both types of cells, ruffled membranes and lamellipodia were abundant at the leading edges of migrating cells and cell processes underwent retraction at the trailing edges. At field strengths of above 10 V/cm, evidence of cellular damage (manifested by cellular rounding and detachment), attributable to the electric field treatment, was observed after 4 hours. These preliminary results suggest that galvanotaxic responses could be exploited clinically in the enhancement of corneal wound healing.

Effects of electric fields on cytoskeleton of corneal stromal fibroblasts.

Low-level, steady electric fields (6-10 volts/cm) stimulated cultured corneal stromal fibroblasts to undergo directional orientation and translocation. The orientative movements (galvanotropism) consisted of somatic elongation of the cells into spindle shapes along an imaginary axis perpendicular to the field; the cathodal edge of the cell underwent retraction, while the anodal edge and the longitudinal ends developed ruffled membranes and lamellipodia. The translocational movements (galvanotaxis) consisted of directed migration of the cells towards the anode. While most actin-containing stress fibers became aligned along the long axes of the elongated fibroblasts (with distal ends of the stress fibers terminating at the longitudinal extremes of the cells), some were aligned towards the anodal direction (with distal terminations inside ruffled membranes and lamellipodia on the leading anodal edge of cells). The distal ends of stress fibers were associated with discrete foci of vinculin, ie, focal indicators of cell-to-substrate adhesion; these foci were abundant at the longitudinal ends and at the anodal edge of the elongated cells. The observed cytoskeletal changes are consistent with an active, rather than passive, directed migration of stromal fibroblasts in response to constant electric fields.

Search for cyclotron resonance in cells in vitro.

There are a number of reports of the plasma membrane transport of Ca2+ in biological systems being enhanced by low frequency electromagnetic fields (EMF), including reports that the enhancement involves a resonance-type response at the cyclotron frequency for Ca2+ ions for geomagnetic values of the magnetic field. Using the fluorescent probe fura2, we find no evidence for changes in cytosolic calcium concentration in BALB/c3T3, L929, V-79, and ROS, a rat osteosarcoma cell line, at the application of both resonant and nonresonant EMF.

Experiments on the Interaction of Electromagnetic Fields with Mammalian Systems.

The results of a series of measurements on the interaction of electromagnetic fields with cell ensembles in vitro are described. The measurements include the effect of fields on population doubling time, 3H-TdR uptake, membrane transport, and galvanotropism. No measureable effect of AC fields was found, and in particular no evidence was found for a cyclotron-resonant mechanism in the ion transport across cell membranes.

Comments on the use of electromagnetic fields in biological studies.

For biological or cellular experiments using electromagnetic fields it is essential that the parameters defining the field be accurately specified if the results are to be meaningful and are to be compared with the same experiment conducted in a different laboratory. The interaction of living systems with electric and magnetic fields can come only through forces exerted on the charges on the system. If the charges are stationary the only origin of the force is the electric field. The electric field may be established by charge distributions, as in "capacitative plate" experiments, or by time-varying magnetic fields consists of a pair of coaxial coils each of equal radius and separated by a distance about equal to the radius. The electric field induced by a varying current in such a pair of coils varies both in space and in time. The field is always zero on the axis of symmetry, and increases to a maximum near the radius of the coils. The strength is proportional to the time-rat-of-change of the current in the coil, which depends not only on the amplitude and shape of the voltage pulse applied to the coil but also on the resistance and inductance of the coil. The purpose of this note is to describe how the important physical parameters may be determined.

Electromagnetic fields in biological studies.

For biological or cellular experiments using electromagnetic fields, it is essential that the parameters defining the field be carefully specified if the results are to be meaningful and are to be compared with the same experiment conducted in a different laboratory. The interaction of living systems with electric and magnetic fields can come only through forces exerted on the charges on the system. If the charges are stationary, the only origin of the force is the electric field. This electric field may be established by charge distributions, as in "capacitive plate" experiments, or by time-varying magnetic fields. A geometry commonly used to produce time-varying magnetic fields consists of a pair of coaxial coils, each of equal radius and separated by a distance often equal to the radius. The electric field induced by a varying current in such a pair of coils varies both in space and in time. The electric field strength is zero on the axis of symmetry, and increases to a maximum near the radius of the coils. The strength is proportional to the time rate of change of the current in the coil, which depends not only on the amplitude and shape of the voltage pulse applied to the coil but also on the resistance and inductance of the coil. The purpose of this article is to describe how these important physical parameters may be determined for both geometries.

Motility of mouse fibroblasts in tissue culture.

The growth and motion of mouse L-cells in vitro have been studied by means of time-lapse photography. In particular, the mitotic period and the motility, defined in terms of [R2], the mean square displacement of an ensemble of cells, have been measured as a function of temperature. The motility is a function of the phase of the cell cycle. For approximately the first one-eighth of the mitotic period the motility is well described as a random walk with persistence, the duration of the persistence being determined by the time of extension of the filopodic spindle. The temperature dependence of the diffusion constant follows the Arrhenius factor. The mitotic period, which varies exponentially as (1/T), exhibits a large variance, and the time difference in replication of daughter pairs follows approximately a Poisson distribution with a mean difference of 138 min at T = 37 degrees C. There is no evidence of mirror symmetry in the motion of daughter pairs for fibroblast cells plated in vitro in Corning tissue culture flasks.

Radioresistance of mongolian gerbils. Fast neutrons.

Seventy-six 8 week old Mongolian gerbils were exposed to acute, whole-body fast neutrons produced by The University of Michigan 83-in. cyclotron. Groups of seven or eigth gerbils were given doses between 485 and 881 rad at 25 rad per minute. The LD 50/30 determined by probit analysis was 750 rad, with 95 per cent fiducial limits of 733 and 776. For the 50 per cent mortality level, an r.b.e. of fast neutrons compared with cobalt-60 of 1-45 was determined. For the same end-point, the r.b.e. for fast neutrons compared with X-rays is 1-33. Mortality data, body-weight and microhaematocrit changes are discussed.