Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.     

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells.     

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).     

Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing.

Transepithelial impedance of Madin-Darby canine kidney cell layers is measured by a new instrumental method, referred to as electric cell-substrate impedance sensing. In this method, cells are cultured on small evaporated gold electrodes, and the impedance is measured in the frequency range 20-50,000 Hz by a small probing current. A model for impedance analysis of epithelial cells measured by this method is developed. The model considers three different pathways for the current flowing from the electrode through the cell layer: (1) in through the basal and out through the apical membrane, (2) in through the lateral and out through the apical membrane, and (3) between the cells through the paracellular space. By comparing model calculation with experimental impedance data, several morphological and cellular parameters can be determined: (1) the resistivity of the cell layer, (2) the average distance between the basal cell surface and substratum, and (3) the capacitance of apical, basal, and lateral cell membranes. This model is used to analyze impedance changes on removal of Ca2+ from confluent Mardin-Darby canine kidney cell layers. The method shows that reduction of Ca2+ concentration causes junction resistance between cells to drop and the distance between the basal cell surface and substratum to increase.     

Characterization of three-dimensional tissue cultures using electrical impedance spectroscopy

Electrical impedance spectroscopy was used to characterize the cell environment of multilayered cell cultures (MCCs), a culture system in which cells are grown on a permeable support membrane to form a thick disc of cells with tumor-like properties. Cultures were grown using SiHa tumor cells as well as V79 wild-type cells and V79/DOX cells cultivated to exhibit multidrug resistance. Electrical impedance measurements were made on MCCs over a frequency range of 0. 1 kHz to 1 MHz. Data analysis using a simple electrical model for the cell environment yielded estimates for parameters related to the intra- and extracellular resistance and net membrane capacitance, which were then related to MCC thickness. The extracellular fraction and tortuosity of the MCCs were determined in separate experiments where the rate of diffusion and the equilibrium level of C14-inulin, which does not penetrate the cell membrane, was measured within MCCs. Impedance measurements predicted the barrier to diffusion posed by the extracellular space of MCCs to be roughly two times greater than that inferred from the C14-inulin experiments. However, the relative ranking of the three cell types used to grow MCCs was similar for the two methods. Results indicate that impedance spectroscopy is well suited for use in characterizing the MCC cell environment, offering a fast, nondestructive method for monitoring cell culture growth and integrity.     

Membrane Operational Impedance Spectra in Chara corallina Estimated by Laplace Transforms Analysis

The membrane operational impedance spectrum of Chara corallina Klein ex Willd. (R. Brown) cells is investigated using Laplace transform analysis. The spectrum changes with both amplitude and sign of the electrical stimulation when time- and voltage-dependent K⁺ channels contribute to the membrane conductance. We compare the advantages and disadvantage of this technique for studying membrane impedance with those of the alternating current method and the white noise method.     

The effect of dendritic voltage-gated conductances on the neuronal impedance: a quantitative model

Neuronal impedance characterizes the magnitude and timing of the subthreshold response of a neuron to oscillatory input at a given frequency. It is known to be influenced by both the morphology of the neuron and the presence of voltage-gated conductances in the cell membrane. Most existing theoretical accounts of neuronal impedance considered the effects of voltage-gated conductances but neglected the spatial extent of the cell, while others examined spatially extended dendrites with a passive or spatially uniform quasi-active membrane. We derived an explicit mathematical expression for the somatic input impedance of a model neuron consisting of a somatic compartment coupled to an infinite dendritic cable which contained voltage-gated conductances, in the more general case of non-uniform dendritic membrane potential. The validity and generality of this model was verified through computer simulations of various model neurons. The analytical model was then applied to the analysis of experimental data from real CA1 pyramidal neurons. The model confirmed that the biophysical properties and predominantly dendritic localization of the hyperpolarization-activated cation current I (h) were important determinants of the impedance profile, but also predicted a significant contribution from a depolarization-activated fast inward current. Our calculations also implicated the interaction of I (h) with amplifying currents as the main factor governing the shape of the impedance-frequency profile in two types of hippocampal interneuron. Our results provide not only a theoretical advance in our understanding of the frequency-dependent behavior of nerve cells, but also a practical tool for the identification of candidate mechanisms that determine neuronal response properties.     

Investigation of the nature of electrical responses of horizontal cells of the fish retina

On the basis of the syncytial structure of the layer of horizontal cells of the fish retina, a method is developed which effectively shifts the membrane potential of cells by means of an electrical current. It is shown that the response of L-type horizontal cells to light and electrical stimulation of the retina is reversed when the membrane of the horizontal cells is depolarized by a direct current. The equilibrium potential of the cells was near the zero level. Consequently, the depolarization response of the horizontal cells to disconnection of the light and to electrical stimulation of the retina is an excitatory postsynaptic potential, whereas hyperpolarization of the horizontal cells to light is a decrease of this potential. It is shown that the membrane of fish horizontal cells have pronounced nonlinear properties: in the case of strong depolarization and especially in the case of hyperpolarization its impedance drops markedly. The latter probably occurs due to an increase of the permeability of the nonsynaptic membrane of the horizintal cells for K⁺. This can also explain the decrease of membrane impedance during the hyperpolarization response of the horizontal cells to bright light. The available data indicate the presence of regenerative properties of the membrane of horizontal cells.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 3, No. 1, pp. 89–98, January–February, 1971.     

Frequency-dependent membrane impedance inChara corallina estimated by Fourier analysis

Summary The white noise method of measuring membrane impedance has been applied to internodal cells ofChara corallina. Fourier analysis of a white noise transmembrane current signal and the voltage response has been used to obtain the frequency-dependent impedance of the in-series combination of the plasmalemma and tonoplast membranes. The results are similar to those of other workers who have measured membrane impedances by different techniques. At very low frequencies the equivalent capacitance of the membrane treated as an RC-circuit becomes negative, indicating a pseudoinductive effect.Membrane impedance has been measured over a range of pH values from pH 5.2 to pH 11; impedance magnitude reaches a maximum at pH 7. At interesting effect of fusicoccin at pH 11 has been observed, in which a decrease in membrane conductance occurs simultaneously with a small hyperpolarization of membrane PD.     

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells.

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.     

Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.     

Depolarization of the neuronal membrane caused by Co-transport of taurine and sodium

C 1300 neuroblastoma cells were cultured and used to study the effect of sodium dependent taurine transport on the membrane potential. Measuring net accumulation of taurine and the depolarization caused by externally applied taurine, we found both processes become active at an external concentration of taurine of 1 mM or more. Net accumulation had K_m of 13 mM and a V_max of 126 nmol × mg of protein^–1×min^–1. The taurine induced depolarization of the neuroblastoma cell was parallelled by a 25 per cent decrease in its membrane impedance. The transport of taurine, the depolarization caused by taurine and the effect of taurine on the membrane impedance, all, had a similar dependence on the external sodium concentration. Our results on the depolarizing cotransport between taurine and sodium at the neuronal membrane, may illustrate an additional mechanism for the control of the electrical activity of neuronal cells.     

Mitochondrial Membrane Studies Using Impedance Spectroscopy with Parallel pH Monitoring

A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample’s complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.     

Phase tracking: an improved phase detection technique for cell membrane capacitance measurements.

We describe here a technique called phase tracking that greatly improves the accuracy of measurements of the membrane capacitance of single cells. We have modified the original phase detection technique to include a method for creating calibrated changes in the resistance in series with the cell. This provides a method to automate the adjustment of the phase detector to the appropriate phase angle for measuring membrane capacitance. The phase determination depends only on the cell's electrical parameters and does not require matching of the cell impedance with that of the slow capacitance cancellation circuitry of the patch-clamp amplifier. We show here that phase tracking can accurately locate the phase of the capacitance signal and can keep the detector aligned with this signal during measurements of exocytosis in mast cells, irrespective of the large drifts which occur in cell membrane resistance, membrane capacitance, or series resistance. The phase tracking technique is a valuable tool for quantifying exocytosis and endocytosis in single cells.     

Impedance analysis of renal vascular smooth muscle cells

Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance (Z_cal) based on the model agreed well with the experimental measurement (Z_exp), and the percentage error defined as |(Z_cal – Z_exp)/Z_exp| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin     

The Action Potentials and Currents on the I-V Plane in the Molluscan Neuron

The action potentials and the corresponding transmembrane currents, directly recorded in the F1 neuron of Helix aspersa by the Self-clamp Technique, were plotted on the I-V plane to represent the real electrical cycle of the cell membrane during activity. The membrane electrical cycle, experimentally obtained, agreed in several aspects with a similar cycle obtained from calculated data on the giant axon of Loligo, but not for the sign, with the consequence of a different localization, as far as voltage and time are concerned, of the negative impedance period. The negative impedance proved to be −614 ± 181 Ω cm² and corresponded to the late phase of the repolarization after the action potential peak. A constant positive impedance was found of 522 ± 131 Ω cm² during the ascending tract of the action potential. These two results are in contrast with previous analyses. The simultaneous availability of the conjugate voltage and current directly measured signals led to the immediate representation of the membrane total conductance in its real time course during activity, in agreement with the Hodgkin and Huxley predictive model. The peak conductance was 1.9 ± 0.7 mmho/cm² in this preparation. The electrical work spent to sustain a single active event proved to be 70 ± 19 nJ/cm². A vectorial representation of the membrane electrical activity is proposed to describe analytically the characteristic behaviour of excitable cells, as well as a new method that utilizes the only action potential to measure the threshold potential in spontaneously discharging cells. The proposed new experimental protocol, based on the use of the Self-clamp Technique, proved to be faster, easier, more productive when compared with the conventional methods; it could be used advantageously in the electrophysiological studies on excitable cells both to define the basic conditions of the investigated preparation and to directly evaluate the effects of subsequent pharmacological stimulations.     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

Impedance of Membrane and Myoplasm during the Action Potential of Frog Muscle

The transient imbalance of a Wheatstone bridge was used to estimate the changes in both membrane and myoplasmic impedance that occurred as an action potential propagated over a 1 mm length of a frog muscle fiber. It was also used to estimate the changes in the membrane impedance alone. Alterations in myoplasmic impedance that might have been predicted were not found.     

Effect of cell electroporation on the conductivity of a cell suspension

An increased permeability of a cell membrane during the application of high-voltage pulses results in increased transmembrane transport of molecules that otherwise cannot enter the cell. Increased permeability of a cell membrane is accompanied by increased membrane conductivity; thus, by measuring electric conductivity the extent of permeabilized tissue could be monitored in real time. In this article the effect of cell electroporation caused by high-voltage pulses on the conductivity of a cell suspension was studied by current-voltage measurements during and impedance measurement before and after the pulse application. At the same time the percentage of permeabilized and survived cells was determined and the extent of osmotic swelling measured. For a train of eight pulses a transient increase in conductivity of a cell suspension was obtained above permeabilization threshold in low- and high-conductive medium with complete relaxation in <1 s. Total conductivity changes and impedance measurements showed substantial changes in conductivity due to the ion efflux in low-conductive medium and colloid-osmotic swelling in both media. Our results show that by measuring electric conductivity during the pulses we can detect limit permeabilization threshold but not directly permeabilization level, whereas impedance measurements in seconds after the pulse application are not suitable.     

Computer simulation of the effect of the nodal gap resistance on ionic current measurements in the Ranvier node membrane.

Results are presented of a computer simulation of the effect of the irreducible resistance introduced by the nodal gap, in series with the impedance of the axon membrane. A clamp potential is applied to a structure modeled as an electric circuit composed of a resistance in series with the membrane impedance, and modified nerve equations describing membrane currents are solved to predict the effect of nodal series resistance on these currents. These studies reveal changes in the absolute values and kinetics of the ionic currents (errors greater than 10-20%) for selected values of series resistance.