Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.     

Intracellular Na+ activity as a function of Na+ transport rate across a tight epithelium.

Liquid Na+ resin microelectrodes were used to measure intracellular Na+ activities (alpha iNa+) in the mammalian tight epithelium, rabbit urinary bladder. alpha iNa+ averaged 7 +/- 1 mM and was independent of Na+ transport rate over the range of 2 to 8 muA/muF. (1 mF congruent to 1 cm2 apical membrane area). After Na+ loading the cells the Na+ pump in the basolateral membrane was measurably electrogenic. A method for shielding the Na+-sensitive microelectrodes is described which increases the response time and eliminates an electrical shunting artifact.     

Differences in membrane properties between unfertilised and fertilised single rabbit oocytes demonstrated by electro-rotation. Comparison with cells from early embryos

The apparent membrane capacity of tubular rabbit oocytes increases from 1.7-2.0 microF/cm2 before fertilisation to 3.7-4.0 microF/cm2 after fertilisation. The membrane conductivity measured on single cells was also increased by fertilisation from less than 1 mS/cm2 to 14 mS/cm2. Cells obtained from 2-, 4- or 8-cell embryos exhibited intermediate values of membrane capacity (2.3-2.8 microF/cm2) and conductivity (5-22 mS/cm2). The values quoted are those effective between 1 and 10 kHz, the frequency of the rotating field used. The large apparent capacities are probably due to the presence of structures such as microvilli which cause the actual membrane area to exceed the smooth sphere area. It must be assumed that these structures change in form or number on fertilisation, and that they persist in embryos, at least up to the 8-cell stage. No difference was apparent between cells fertilised in vitro or in vivo. Comparison of the above zona-free data with measurements on zona-complete oocytes indicate how fertilised and unfertilised rabbit eggs may be distinguished from one another, even in the presence of the zona pellucida.     

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6+/-0.9 to 9.1+/-0.5 mF/m(2) 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.     

Dielectrophoretic spectra of single cells determined by feedback-controlled levitation.

In this paper we have utilized the principle of dielectrophoresis (DEP) to develop an apparatus to stably levitate single biological cells using a digital feedback control scheme. Using this apparatus, the positive DEP spectra of both Canola plant protoplast and ligament fibroblast cells have been measured over a wide range of frequencies (1 kHz to 50 MHz) and suspending medium conductivities (11-800 muS/cm). The experimental data thus obtained have been interpreted in terms of a simple spherical cell model. Furthermore, utilizing such a model, we have shown that various cellular parameters of interest can be readily obtained from the measured DEP levitation spectrum. Specifically, the effective membrane capacitance of single cells has been determined. Values of 0.47 +/- 0.03 muF/cm2 for Canola protoplasts and 1.52 +/- 0.26 muF/cm2 for ligament fibroblasts thus obtained are consistent with those determined by other existing electrical methods.     

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.     

Electrophysiological properties of tissue cultured heart cells grown in a linear array.

Embryonic chick heart cells were grown in tissue culture on an oriented substrate (channels cut in an agar coated slide), so that they formed narrow(5-100mu) strands of arbitrary length. The electrical properties of these strands were examined using intracellular microelectrodes. ac and dc cable studies were performed to determine the passive cable parameters. Quantitative histology, using light and electronmicroscopy, permitted calculation of intrinsic capacitances and resistivities. Electrical coupling between polarizing and recording electrodes was ubiquitous, falling off exponentially with distance. It was concluded that individual cells were electrically connected, since coupling was observed at distances greater than 3 mm, and the maximum cell length was estimated to be less that 300 mu. The strands were usually spontaneously active, with phase 4 depolarization (pacemaker potential) occurring almost simultaneously in all cells of a strand. The passive electrical properties determined during phase 4 were: core resistivity (cytoplasm plus cell-to-cell resistance), 245 ohm/cm; membrane capacitance, 1.46 muF/CM2. The membrane resistance increased from 16 to 136 kohm/cm2 during phase 4. The space and time constants showed commensurate changes, from 0.95 to 3.2 mm, and from 29 to 269 msec, respectively. The input resistance also increased, from 1.1 to 3.8 Mohm.     

Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis

We have used dielectrophoresis to determine the dielectric properties of human chronic myelogeneous leukaemic (K562) cells during apoptosis (programmed cell death). Our results indicate that K562 cells increase markedly in cytoplasmic conductivity from 0.28 S/m to 0.50 S/m within the first 4 h following treatment with staurosporine, which then lasts beyond 12 h, whilst cell shrinkage increases the capacitance of the membrane from 9.7 mF/m2 to 20 mF/m2. After 24 and 48 h of incubation with staurosporine, multiple sub-populations were detected, highlighted by the dielectric changes that the cell undergoes before death. By comparing these results with those obtained by common apoptosis monitoring techniques Annexin V and TMRE (tetramethylrhodamine ethylester), it is possible to infer the role of ion efflux in the progress of apoptosis. The use of dielectrophoresis for monitoring apoptosis offers a number of benefits as it is both rapid and non-invasive. It can also be used in parallel with other assays in high-throughput screening applications.     

Dielectric properties of yeast cells as determined by electrorotation.

Electrorotational spectra of yeast cells, Saccharomyces cerevisiae strain R XII, were measured over a frequency range of nearly 7 decades. The physical properties of distinct cell parts were simultaneously determined for individual cells by comparison with an electrical two-shell model: The conductivity of the cytoplasm, cell wall and cytoplasmic membrane of living cells were found to be 5.5 mS/cm, 0.1 to more than 0.5 mS/cm and less than 0.25 nS/cm to 4.5 microS/cm, respectively. The conductivity of the cytoplasmic membrane was dependent on the conductivity of the medium. Membrane behaviour is interpreted as an opening of membrane channels when the environment becomes more physiological. The specific membrane capacitance was determined to be 1.1 microF/cm2 and the thickness of the cell wall was calculated as 0.11 micron. Heat treated cells showed an increased membrane conductivity of more than 0.1 microS/cm (at 25 microS/cm medium conductivity) and a drop in cytoplasmic conductivity to between 0.1 and 0.8 mS/cm, depending on the length of time the cells were suspended in low conductivity water (25 microS/cm), indicating a perforation of the membrane. A slightly decreased spinning speed scaling factor for dead cells suggests a modification to the cellular surface, while the principal structure of the cell wall appears to be uneffected. It can be demonstrated by these observations, that cellular electrorotation permits the simultaneous investigation of the different cellular compartments of individual cells in vivo under various environmental conditions.     

Asymmetry currents and admittance in squid axons.

The complex admittance of squid (Loligo pealei) axon was measured rapidly (within 1 s) with pseudo-random small signals and discrete Fourier transform techniques under guarded, "space-clamp" conditions and during suppression of ion conduction. Asymmetry currents were measured by paired step clam pulses of +/-70 mV from a holding potential of -97 mV and gave an apparent capacitance of 0.36 muF/cm2. However, the admittance data showed no change in capacitance at holding potentials from -97 to -67 mV and gave a decrease of 0.07 of 0.15 muF/cm2 at -37 mV. The failure to observe a capacitance increase at low membrane potentials suggests the following possibilities: (a) the asymmetry current is a displacement current that inactivates completely with time, and (b) the asymmetry current is not a displacement current and arises from large signal effects (i.e., delayed nonlinearity in ionic current) on the membrane.     

Electrorotation--a new method for investigating membrane events during thrombocyte activation. Influence of drugs and osmotic pressure

The measurement of the spin of cells in rotating high-frequency electric fields ('electrorotation') make possible the investigation of dielectric membrane properties of single cells. This method was applied to membrane permeability changes accompanying thrombocyte activation and compared with light-scattering data. Describing the dielectric behavior of platelets by a single-shell model and assuming a sufficiently low membrane conductivity of 1 X 10(-7) S/m we found for nonactivated platelets a membrane capacity of 5.5 mF/m2 and the conductivity of the internal medium was estimated to be 0.12 S/m. Upon activation, the electrorotation decreased continuously, with half-times in the range of few minutes. This could be explained assuming a 500-fold increase in membrane conductivity. The application of both local anesthetics and virostatics inhibited the decrease of electrorotation, as did hypertonic osmotic pressure. In all cases this was accompanied by inhibition of platelet aggregation. Hypotonic solutions induced self-aggregation and spontaneous loss of electrorotation. It was concluded that the increase in permeability of the granule membrane is a crucial step in the release reaction and that the electrorotation method is able to detect the incorporation of the granule membranes into the plasma membrane during activation. The advantage of this electrorotation method is that it enables measurements on a single-cell level, thus avoiding interactions between platelets.     

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6±0.9 to 9.1±0.5 mF/m² 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.     

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

Regulation of Ganglioside Metabolism by Phosphorylation and Dephosphorylation

Abstract: Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP-NeuAc:GM1 α2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108-15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST-IV activity to 45% of that of control cells with t_1/2 = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST-IV due to the inhibition of a serine/threonine-specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST-IV, the activity of GalNAc-T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in conjunction with down-regulation of ST-IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a-series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phosphorylation systems.     

Membrane dielectric responses of bufalin-induced apoptosis in HL-60 cells detected by an electrorotation chip

A non-invasive electrorotation (ROT) technique was used to monitor the apoptosis-induced changes in HL-60 cells. The specific membrane capacitance of the cells fell from 15.6 ± 0.9 mF/cm² to 6.4 ± 0.6 mF/cm² after 48 h treatment with 10 nM bufalin, a component of bufadienolides in traditional Chinese medicine, Chan Su. However, the average membrane conductance remained almost constant during the first 24 h of treatment and then increased afterwards. Apoptosis was verified by a DNA fragmentation assay and scanning electron microscopy. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents. Chengjun Huang and Ailiang Chen contributed equally to this work.     

Modification of acetylcholine sensitivity of neuronal membrane by presynaptic stimulation

The action of electrical stimulation of one of the pallial nerves on the sensitivity of the bursting RPa1 neuron of Helix pomatia to acetylcholine (ACh) was investigated. The depolarizing effect of ACh was significantly decreased by presynaptic stimulation. Stimulation leads also to an attenuation of the ACh-induced increase in membrane conductivity. The effect of stimulation on the ACh evoked response of the membrane was reversibly blocked by cold and was completely eliminated after long term incubation of the neuron under "in vitro" conditions.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.