Yukio Doida

Passive electrical properties of cultured murine lymphoblast (L5178Y) with reference to its cytoplasmic membrane, nuclear envelope, and intracellular phases

SummaryDielectric dispersion measurements over a frequency range 0.01–100 MHz were made with the suspensions of a cultured cell line, mouse lymphoma L5178Y, and an attempt to explain the observed dielectric behavior by taking explicitly into consideration the possible involvement of cell nucleus has been presented.The use of a conventional “single-shell” model in which the cell is represented by a homogeneous sphere coated with a thin limiting shell phase did not duplicate the observed dispersion curves, whereas a “double-shell” model in which one additional concentric shell is incorporated into the “single-shell” model gave a much better fit between the observed and the predicted dispersion curves. Based on the latter model, we analyzed the raw data of dielectric measurements to yield a set of plausible electrical parameters for the lymphoma cell:CM≅1.0μF/cm2,CN≅0.4μF/cm2, εk≅300, κc/κa≅0.9, and κk/κc≅0.7. Here,CM andCN are the specific capacities of plasma and nuclear membranes; ε and κ are the dielectric constant and conductivity with subscripta, c andk referring respectively to the extracellular, the cytoplasmic and the karyoplasmic phases.

Oscillations of membrane potential in L cells. I. Basic characteristics.

SummaryThe membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around −15 mV and 37 MΩ, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around −15 and −40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K+, Na+ and Cl− concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated.

Oscillations of membrane potential in L cells. II. Effect of monovalent ion concentrations and conductance changes associated with oscillations.

SummaryOscillation and activated hyperpolarizing responses induced by electrical stimuli (H.A. responses) were studied in large nondividing L cells (giant L cells) under a variety of ionic conditions. When Cl− in the bathing fluid was partially replaced with SO42− at fixed external Na+ and K+ concentrations, the membrane potential depolarized transiently, but recovered to the original potential level after about 10 min. Under such a steady state in a low-Cl− medium, the amplitudes of oscillations and H.A. responses remained almost identical with those in the control medium. On exposure to a low-Na+ medium, both membrane potentials in the resting and hyperpolarized states were slightly hyperpolarized, but the pattern and the amplitude of oscillations and H.A. responses remained much the same. Changes in external K+ concentrations remarkably affected the amplitudes of oscillations and H.A. responses: the amplitudes decreased with increases in external K+ concentration. Calculation of the changes in K+, Na+ and Cl− conductances during oscillations and H.A. responses under these various ionic conditions showed that the change in K+ conductance is the only factor responsible for the oscillation and the H.A. response. The reversal potential for the potential oscillation is about −94 mV under normal conditions, this value being quite close to that of the equilibrium potential of K+. The reversal potentials in various external K+ concentrations satisfied the Nernst equation for a K+ electrode. Valinomycin induced remarkable hyperpolarization of the resting potential, resulting in an inhibition of oscillations. The level of valinomycin-induced hyperpolarization of the resting potential required to inhibit H.A. responses was the same as that of the peak potentials of the oscillation and H.A. response. In the light of these observations, it is concluded that the spontaneous potential oscillation and the H.A. response are caused solely by increase in the K+ conductance of the cell membrane.

Effects of cytochalasin B and local anesthetics on electrical and morphological properties in L cells

Abstract Spontaneous oscillations of membrane potential observed in L cells were inhibited rapidly and reversibly in the presence of cytochalasin B (CB). Sustained hyperpolarization induced by high external Ca 2+ was also depressed by the drug. However, Ca 2+ injection into the cytoplasm elicited a sustained hyperpolarization, even in the presence of CB. These observations strongly suggest that CB inhibits calcium transport system in cell membrane. Morphological alterations associated with the CB treatment were decreased adhesiveness and rounding of the cells, with concomitant changes in surface architecture. Similar changes in electrophysiological and morphological properties were observed in cells treated with local anesthetics. Since such morphological changes induced by CB and local anesthetics were always preceded by electrical changes, it was suggested that the morphological changes are secondary phenomena resulting from inhibition of the Ca 2+ transport.

STUDIES ON SOME RADIATION-INDUCED CHROMOSOME ABERRATIONS IN MAN

It has been reported by Buckton et al. (1), Bender and Gooch (2, 3), Boyd et al. (4), Tough et al. (5), and Sasaki et al. (6) that ionizing radiations induce chromosome aberrations in peripheral leukocytes of man and that the aberrations may persist for years. In the present investigation, chromosome studies were made on peripheral leukocytes taken from persons exposed to atomic bomb radiation more than 17 or 18 years ago in Hiroshima or Nagasaki, from patients under daily radiotherapy after breast cancer surgery, and from radiation personnel who had been exposed to fairly large doses of radiation during a long period of working time. The purposes of the present study are as follows: (1) To determine the types and frequencies of aberrations induced. (2) To find special types of aberrations, if any, which may persist predominantly for a long time and be related to delayed effects of radiation such as carcinogenesis or aging. (3) To test the possibility of developing a new method of biological dosimetry by means of quantitative evaluation of chromosome aberrations.

Cytogenetic Studies on Radioresistant Cells Derived from Mouse Strain L Cells in Cell Culture

Several variants have been obtained from mouse strain L cells in cell culture after repeated treatments with chemical and physical agents such as mitomycin C, 8-azaguanine, and ultraviolet irradiation (1). The radiosensitivity of L cells has been studied by Whitfield and Rixon (2), who observed that radioresistant derivatives could be obtained after a treatment with a single large dose of X-irradiation. On the other hand, Rhynas and Newcombe (3) have obtained some strains resistant to X-ray by repeated exposures of mass cultures of the original L cells. In the present investigation, by repeated y-irradiation of surviving cells on each sequential passage by a method similar to those reported previously (1), a pure line of resistant cells which differed distinctively in chromosome distribution from the original L cells was obtained. These resistant cells could provide a useful tool for genetic and biochemical studies of somatic mammalian cells in vitro, such as in studies of transformation of the type seen in microorganisms. Furthermore, comparison of various genetic characters of radioresistant cells with those of the original L cells could shed light on the nature of radiation effects on mammalian cells. The present paper describes the isolation from the original L cells of cells resistant to -y-rays, and the cytogenetic characters of these resistant cells.