Gadolinium blocks membrane permeabilization induced by nanosecond electric pulses and reduces cell death

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
Bioelectrochemistry (Amsterdam, Netherlands) (Impact Factor: 4.17). 08/2010; 79(1):95-100. DOI: 10.1016/j.bioelechem.2009.12.007
Source: PubMed


It has been widely accepted that nanosecond electric pulses (nsEP) are distinguished from micro- and millisecond duration pulses by their ability to cause intracellular effects and cell death with reduced effects on the cell plasma membrane. However, we found that nsEP-induced cell death is most likely mediated by the plasma membrane disruption. We showed that nsEP can cause long-lasting (minutes) increase in plasma membrane electrical conductance and disrupt electrolyte balance, followed by water uptake, cell swelling and blebbing. These effects of plasma membrane permeabilization could be blocked by Gd(3+) in a dose-dependent manner, with a threshold at sub-micromolar concentrations. Consequently, Gd(3+) protected cells from nsEP-induced cell death, thereby pointing to plasma membrane permeabilization as a likely primary mechanism of lethal cell damage.

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    • "In physiological media, because larger intracellular solutes cannot cross the permeabilized membrane, the intracellular osmolality becomes greater than the extracellular osmolality . This is countered by water influx into the cell, resulting in an increase in cell volume (cell swelling) [37] [38]. The advantage of this approach compared to methods based on fluorescent, impermeant dyes for the study of electropermeabilization induced by nanosecond pulses is that the sensitivity of the latter is limited by the number and size of the pores created. "
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    ABSTRACT: Pulsed electric fields are used to permeabilize cell membranes in biotechnology and the clinic. Although molecular and continuum models provide compelling representations of the mechanisms underlying this phenomenon, a clear structural link between the biomolecular transformations displayed in molecular dynamics (MD) simulations and the micro- and macroscale cellular responses observed in the laboratory has not been established. In this paper, plasma membrane electropermeabilization is characterized by exposing Jurkat T lymphoblasts to pulsed electric fields less than 10ns long (including single pulse exposures), and by monitoring the resulting osmotically driven cell swelling as a function of pulse number and pulse repetition rate. In this way, we reduce the complexity of the experimental system and lay a foundation for gauging the correspondence between measured and simulated values for water and ion transport through electropermeabilized membranes. We find that a single 10 MV/m pulse of 5ns duration produces measurable swelling of Jurkat T lymphoblasts in growth medium, and we estimate from the swelling kinetics the ion and water flux that follows the electropermeabilization of the membrane. From these observations we set boundaries on the net conductance of the permeabilized membrane, and we show how this is consistent with model predictions for the conductance and areal density of nanoelectropulse-induced lipid nanopores.
    Biochimica et Biophysica Acta 04/2013; 1828:1715-1722. DOI:10.1016/j.bbamem.2013.03.007 · 4.66 Impact Factor
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    • "This indicates that nsPEFs target mitochondria membranes with dissipation of ΔΨm as a major factor initiating cell death. This is in contrast to studies using gadolinium concluding that plasma membrane permeability is a likely mechanism of lethal cell damage [29]. However, another important point to note is that intracellular calcium, coming primarily through plasma membranes in N1-S1 cells, significantly potentiates dissipation of ΔΨm and thereby potentiates cell death. "
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    ABSTRACT: It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.
    PLoS ONE 12/2012; 7(12):e51349. DOI:10.1371/journal.pone.0051349 · 3.23 Impact Factor
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    • "T80 (0.5 %) (Wallace et al. 1968) has been implicated in mitochondrial and membrane biogenesis in yeast, a factor that was not investigated in these studies but which may influence both assay outcomes through different mechanisms. The plasma membrane is significantly affected after exposure to nsPEFs (André et al. 2010) measured by propidium iodide uptake across the cell membrane. The trypan blue assay is an assay based on the integrity of the plasma membrane. "
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    ABSTRACT: We investigated the effects of nanosecond pulse electric fields (nsPEFs) on Jurkat and PANC1 cells, which are human carcinoma cell lines, in the presence of Tween 80 (T80) at a concentration of 0.18 % and demonstarted an enhanced killing effect. We used two biological assays to determine cell viability after exposing cells to nsPEFs in the presence of T80 and observed a significant increase in the killing effect of nsPEFs. We did not see a toxic effect of T80 when cells were exposed to surfactant alone. However, we saw a synergistic effect when cells exposed to T80 were combined with the nsPEFs. Increasing the time of exposure for up to 8 h in T80 led to a significant decrease in cell viability when nsPEFs were applied to cells compared to control cells. We also observed cell type-specific swelling in the presence of T80. We suggest that T80 acts as an adjuvant in facilitating the effects of nsPEFs on the cell membrane; however, the limitations of the viability assays were addressed. We conclude that T80 may increase the fragility of the cell membrane, which makes it more susceptible to nsPEF-mediated killing.
    Journal of Membrane Biology 07/2012; 245(10):611-6. DOI:10.1007/s00232-012-9472-0 · 2.46 Impact Factor
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