Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state.

The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, P(p) , in response to an externally applied magnetic force. P(p) is inversely coupled with leaf turgor pressure, P(c) , i.e. at high P(c) values the P(p) values are small and at low P(c) values the P(p) values are high. This relationship between P(c) and P(p) could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (P(c) dropped below ca. 50 kPa), P(p) curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum P(p) value, and during darkness a peak P(p) value is recorded. This reversal of the P(p) curves was completely reversible. Upon watering, the original diurnal P(p) changes were re-established within 2-3 days. Olive trees in the field showed a similar turnover of the shape of the P(p) curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the P(p) curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal P(p) curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental P(p) curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero P(c) .

Single center method: a computational tool for ionization and electronic excitation studies of molecules.

We discuss the recent progress in the development of the single center (SC) method for computation of highly-delocalized discrete and partial photoelectron wave continuous functions of molecules. Basic equations of the SC method are presented, and an efficient scheme for the numerical solution of a system of coupled Hartree-Fock equations for a photoelectron is described. Several illustrative applications of the method to photoionization and electronic excitation processes in diatomic molecules are considered. Thereby, we demonstrate its potential for theoretically studying angularly resolved molecular photoionization processes.

Symmetry-forbidden electronic state interference observed in angularly resolved NO+ (A 1Π) deexcitation spectra of the N*O(2σ(-1)2π(2)) resonance.

Quantum mechanical interference between different pathways in inner-shell resonance excitation-deexcitation spectra is a realization of a double-slit experiment on the atomic scale. If the intermediate inner-shell resonances are of different symmetries, this interference is symmetry forbidden in the solid-angle-averaged or magic-angle-recorded deexcitation spectra. It has, however, been suggested that interference may by observable in off-magic-angle-recorded spectra. Here, we prove this interference in angularly resolved deexcitation spectra of the 2σ(-1)2π(2)(2Δ,2Σ±) resonances of N*O by a quantitative comparison between ab initio calculations and experiment.

Neutral dissociation of the I, I', and I" vibronic progressions of O2.

It is suggested that the main mechanism for neutral dissociation of the I, I('), and I(") vibronic progressions in O(2) is due to their interaction with the vibrational continuum of the 1pi(u) (-1)(A (2)Pi(u))3ssigma(g) (3)Pi(u)(v(epsilon)) Rydberg state (J state) leading to the formation of the O(2p(4) (3)P)+O( *)(2p(3)((4)S)3s (3)S) fragments. In order to justify this, the O I 2p(3)((4)S)3s (3)S-->2p(4) (3)P fluorescence emission cross section following the neutral dissociation of the O(2) 1pi(u) (-1)(a (4)Pi(u))4ssigma(g)/3ddelta(g)/3dsigma(g) (3)Pi(u)(v) Rydberg states is simulated in the exciting-photon energy range of 14.636-16.105 eV. The results of high-resolution measurements (H. Liebel et al., J. Phys. B 34, 2581 (2001)) can be reproduced if a small adjustment of the computed potential curve of the J state is applied. Non-Franck-Condon resonant intensity distributions of the I, I('), and I(") progressions observed in the experiment are qualitatively explained by the presence of the O(2) 1pi(g) (-1)(X (2)Pi(g))npsigma(u)/nfsigma(u)/nfdelta(u) (3)Pi(u) perturber states. Present calculations allow to decide between two different assignments of the I, I('), and I(") states available in literature.

Effects on capacitance by overexpression of membrane proteins.

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4)channels/mum(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C(m)). The C(m) increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C(m) of 1.15+/-0.08 microF/cm(2) in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79+/-0.02 microF/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C(m) (0.85+/-0.07 microF/cm(2)) as compared to non-expressing control cells (0.70+/-0.03 microF/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.

A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions.

Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB(-)) and [W(2)(CO)(10)(S(2)CH)](-), the putative lipophilic anion B(CF(3))(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB(-) and B(CF(3))(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB(-) and B(CF(3))(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.

Mechanisms of electrically mediated cytosolic Ca2+ transients in aequorin-transformed tobacco cells.

Cytosolic Ca(2+) changes induced by electric field pulses of 50-micros duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca(2+)-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca(2+) concentration reaching a peak value within <100-200 ms. Peaking of [Ca(2+)](cyt) was followed by a biphasic decay due to removal of Ca(2+) (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of approximately 0.5 and 3-5 s, respectively. Experiments with various external Ca(2+) concentrations and conductivities showed that the fast decay arises from Ca(2+) fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca(2+) fluxes through the tonoplast. The amplitude of the [Ca(2+)](cyt) transients increased with increasing field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical field strength of approximately 450 V/cm, whereas breakdown of the tonoplast required approximately 580 V/cm. The above findings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca(2+) and presumably other vacuolar ingredients.

Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system.

Giant HEK293 cells of 30-65 microm in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1+/-0.1 microF/cm2 for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 microm) was found to be between 12.8 and 16.1 microS/cm2, which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system.

Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation.

Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane (probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C(m) (microF/cm2). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C(C) (pF) in strongly hypotonic solutions.

Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation.

The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice.

Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion.

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways.

Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1-2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10-15 min. RVD also occurred in strongly hypotonic 100 mOsm solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose, galactose, inositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity and volume-dependent solute permeabilities. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.

Raman regime energy dependence of alignment and orientation of KrII states populated by resonant Auger effect.

The energy dependencies of alignment parameters A20 for KrII 4p(4)5p states after the Auger decay of the KrI 3d(9)np resonances were investigated theoretically and experimentally for the first time in the Raman regime with the bandwidth of the exciting radiation (deltaE(FWHM)=20 meV) smaller than the natural width of the resonances (Gamma approximately 80 meV). The observed energy dependence is due to the in-terference between the different resonance channels and the direct photoionization channel. A strong energy dependence for both the orientation parameter O10 and the photoelectron angular distribution parameter beta(el) is also predicted.

Intracellular delivery of trehalose into mammalian cells by electropermeabilization.

The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.

Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation.

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.

Trehalose improves survival of electrotransfected mammalian cells.

BACKGROUND: Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. MATERIALS AND METHODS: Various mammalian cell lines were electropermeabilized by single exponentially decaying electric pulses of few kV/cm strength and of several-microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI-uptake, GFP gene expression, transfection yield, and short- and long-term viability were analyzed by flow cytometry and electronic cell counting. RESULTS: The substitution of inositol by trehalose in pulse media protected cells against field-induced cell lysis. The protection effect saturated at about 40-50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. CONCLUSIONS: We demonstrated that trehalose-substituted media are superior to standard trehalose-free pulse media for improving cell survival and achieving higher electrotransfection efficiency.

Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration.

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to approximately 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigmae is smaller than that of the cytosol sigmai. The stretching force vanishes when sigmae is equal to or larger than sigmai. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).

Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.

The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)(5)CN](-) across the plasma membrane of mammalian cells. The adsorption of [W(CO)(5)CN](-) was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC(50) for the effect of phloretin on the transport parameters of the lipophilic ion was approximately 10 microM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.

Effect of fluorine substitution on the interaction of lipophilic ions with the plasma membrane of mammalian cells.

The effects of the anionic tungsten carbonyl complex [W(CO)(5)SC(6)H(5)](-) and its fluorinated analog [W(CO)(5)SC(6)F(5)](-) on the electrical properties of the plasma membrane of mouse myeloma cells were studied by the single-cell electrorotation technique. At micromolar concentrations, both compounds gave rise to an additional antifield peak in the rotational spectra of cells, indicating that the plasma membrane displayed a strong dielectric dispersion. This means that both tungsten derivatives act as lipophilic ions that are able to introduce large amounts of mobile charges into the plasma membrane. The analysis of the rotational spectra allowed the evaluation not only of the passive electric properties of the plasma membrane and cytoplasm, but also of the ion transport parameters, such as the surface concentration, partition coefficient, and translocation rate constant of the lipophilic anions dissolved in the plasma membrane. Comparison of the membrane transport parameters for the two anions showed that the fluorine-substituted analog was more lipophilic, but its translocation across the plasma membrane was slower by at least one order of magnitude than that of the parent hydrogenated anion.

Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents.

The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca(2+) sequestration) in the vicinity of the cell membrane.