Roland Glaser

Dielectric spectroscopy of human erythrocytes: investigations under the influence of nystatin

When placed in rotating electric fields red blood cells show a typical electrorotation spectrum with antifield rotation in the lower and cofield rotation in the higher frequency range. Assuming a spherical cell geometry, however, dielectrical parameters were obtained that differ from those measured by independent methods. Dielectrophoresis and, in particular, electrorotation yielded lower membrane capacitance values than expected. Introduction of an ellipsoidal model with an axis ratio of 1:2 allowed a description that proved to be consistent with dielectrophoresis and electrorotation data. For control cells an internal conductivity of 0.535 S/m, a specific membrane capacitance of 0.82 x 10(-2) F/m2, and a specific conductance of 480 S/m2 were obtained. The first characteristic frequency (frequency of fastest antifield rotation) and the related rotation speed can be measured quite quickly by means of a compensation method. Thus it was possible to follow changes of dielectric properties on individual cells after nystatin application. Ionophore-membrane interaction caused cell shrinkage in parallel to a decrease of the first characteristic frequency and rotation speed. Analysis of data revealed a decrease of the internal conductivity that is not only caused by ion loss but also, to a large extent, by a strong increase of hindrance because of shrinkage. Ionophore-induced membrane permeabilities can be calculated from volume decrease as well as from electrorotational data. In no case can these permeabilities count for the high membrane-AC conductivity that is attributed to the band-3 anion exchanging protein. The membrane-AC conductance was found not to be decreased for cells in Donnan equilibrium, which had leaked out almost completely.

Thermal mechanisms of interaction of radiofrequency energy with biological systems with relevance to exposure guidelines.

This article reviews thermal mechanisms of interaction between radiofrequency (RF) fields and biological systems, focusing on theoretical frameworks that are of potential use in setting guidelines for human exposure to RF energy. Several classes of thermal mechanisms are reviewed that depend on the temperature increase or rate of temperature increase and the relevant dosimetric considerations associated with these mechanisms. In addition, attention is drawn to possible molecular and physiological reactions that could be induced by temperature elevations below 0.1 degrees, which are normal physiological responses to heat, and to the so-called microwave auditory effect, which is a physiologically trivial effect resulting from thermally-induced acoustic stimuli. It is suggested that some reported “nonthermal” effects of RF energy may be thermal in nature; also that subtle thermal effects from RF energy exist but have no consequence to health or safety. It is proposed that future revisions of exposure guidelines make more explicit use of thermal models and empirical data on thermal effects in quantifying potential hazards of RF fields.

The shape of red blood cells as a function of membrane potential and temperature.

SummaryIt is well known that a pH shift of the outside medium from 5 to 9 produces a shape transformation of washed human red blood cells from stomatocytes to echinocytes in isotonic salt solutions. In addition, a stomatocytogenic effect is demonstrated here due to solutions of low ionic strength (below 70mm). An analysis of the true cell state in these situations, proved by measurements of predicted volume changes, indicates a good correlation between transmembrane potential and cell shape. The fact that amphotericin B acts as echinocytogenic agent in low ionic strength medium at pH 7.4 but not at pH 5.1 underlines this explanation. Therefore, a transmembrane potential positive inside produces stomatocytes, slightly negative inside (below−10 mV), normocytes, and strongly negative, echinocytes. The temperature dependence of this process underlines the rigidity-pattern hypothesis of red blood cell shape (Glaser & Leitmannová, 1975, 1977).

Rotation of dielectrics in a rotating electric high-frequency field. Model experiments and theoretical explanation of the rotation effect of living cells

Model experiments are carried out to clarify the mechanism of rotation of living cells in a rotating electric field. According to classical investigations of the rotation of macroscopic bodies in external fields, the rotation of spherical glass vessels or metal cylinder filled with electrolyte solutions was investigated. The relation of the calculations of Lertes (1921a,b) to the recent paper of Arnold and Zimmerman (1982) and our new derivations lead to equations explaining the rotation of objects. The results are compared with measurements using mesophyll protoplasts and data from the literature.

Echinocyte formation induced by potential changes of human red blood cells.

SummaryIn isotonic 30mm NaCl-saccharose solution, human red blood cells with intact membrane and normal inside ionic content (C-state) indicate a transmembrane potential between +30 mV (at pH 7.4) and +46 mV (at pH 5.1). After treatment with amphotericin B or nystatin as ionophores, a Donnan equilibrium (D-state) will be reached with the same potential at pH 5.1 but a sharp drop down to −20 mV will occur at pH 7.4. Concerning the erythrocyte shape at these states, a stomatocyteechinocyte transformation takes place, in correlation with the potential shift. Stomatocytes formed at ΔΨ>+25 mV, echinocytes at ΔΨ<+25 mV. At potentials lower than +5 mV, no further effect can be observed. This process is reversible. Neuraminidase treatment as well as outside EDTA do not influence this process significantly. Human serum albumin in concentrations of 2% stabilizes the stomatocytes.

The electric potential profile across the erythrocyte membrane

Abstract The membrane potential profile of erythrocytes is calculated on the basis of realistic data on the electric charges of the glycocalyx, the spectrin layer as well as of the phosphatidyl serine molecules. Various stationary and quasi-stationary osmotic states of erythrocytes are considered. The calculations are performed by numerical integration of the nonlinear Poisson-Boltzmann equation. It is shown that the potential profile is strongly influenced by the negative charges of phosphatidyl serine at the inner membrane surface. For all osmotic states a negative inner surface potential of more than 60 mV was calculated. The basic model is extended by incorporation of a specific binding of the cations calcium, magnesium and sodium to phosphatidyl serine as well as by consideration of the finite volumes of the ions of the electrolyte. Both effects have only a weak influence on the membrane potential profile of erythrocytes.

Membrane bending energy in relation to bilayer couples concept of red blood cell shape transformations

Abstract A simple four parameter geometrical model is introduced to approximately simulate the axisymmetrically shaped red blood cells. Possible shapes are calculated according to the requirement that the cell volume and areas of the two constituent monolayers of the membrane are constant during cell shape transformations. The intervals for the geometrical parameters are determined within which the cell can have a series of rotationally symmetrical shapes. The cell shape at given values of the above three cell properties is obtained by finding the minimum value of the membrane bending energy. The presented picture of red blood cell shape transformations is shown to be in agreement with the concept of bending energy as well as with the bilayer couples concept.

Influence of surface charge and transmembrane potential on rubidium-86 efflux of human red blood cells

SummaryThe dependence of the rate constant of Rb+ efflux on extracellular cation concentration was measured. At low ionic strengths Rb+ efflux increased strongly. Permeability coefficients were calculated from the rate constants measured, using the Goldman flux equation, with and without making allowance for surface potentials. Only when allowance was made for surface potentials and the associated differences beween ion concentrations in the bulk solutions and at the membrane surface, the permeability coefficient remained constant. Best agreement between experimental data and theoretically calculated values was obtained when an interior surface potential of − 110 mV was assumed.When the surface charge of erythrocytes is reduced by neuraminidase, the rate constants for Rb+ efflux decreased, indicating a significant influence of surface potential.

Electrorotation of lymphocytes—The influence of membrane events and nucleus

Electrorotation—the spin of cells in rotating high frequency electric fields—has been used to investigate properties of human peripheral blood lymphocytes. The rotation spectra of lymphocytes deviate from those of single shell spheres. The deviations are caused by the electrical properties of the nucleus in the cell interior.Electrorotation allows the distinction between successfully stimulated lymphocytes and unstimulated cells after application of concanavalin A. Notwithstanding the fact that only a proportion of the cells will be mitogenically stimulated we detected an enhanced cell membrane conductivity for the whole cell population immediately after the addition of mitogen.

278 - Osmotic states of the red blood cells

Abstract 1. Permeative properties of the red blood cell membrane are utilized for an introduction of three osmotic states of the model cell with respect to equilibration of certain cell solution constituents across the membrane equilibration of water (W state), equilibration of water and chloride (C state), and equilibration of water, chloride and cations (D state). 2. The model cell introduced consists of the semipermeable membrane which separates the buffered external solution composed of water, univalent electrolyte, and sucrose from the internal hemoglobin and electrolyte solution. Three sets of equations each describing corresponding osmotic state are given. Certain physicochemical properties of the hemoglobin solution are taken into account. The inner pH is assumed to be established according to the chloride distribution across the membrane. 3. The volume and electric potential difference of the model cell are calculated for each osmotic state at different ratios of sucrose and electrolyte concentration of external solutions. Results are used for an analysis of general osmotic properties and lysis of red cells. 4. It is shown how cells are expected to behave if exposed to conditions with different osmotic pressure, composition of external solutions, or temperature. The importance of temperature effect and rate of performing osmotic experiments on the existence of the W state is pointed out. 5. Different experimental procedures which cause an increase in the membrane cation premeability are discussed in terms of the transition from the C to D state. It is indicated that after the membrane modification in the cases discussed osmotic accomodation of cells is governed by the net-chloride diffusion.