Recruitment of the intracellular Ca2+ by ultrashort electric stimuli: The impact of pulse duration

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. Electronic address: .
Cell calcium (Impact Factor: 3.51). 06/2013; 54(3). DOI: 10.1016/j.ceca.2013.05.008
Source: PubMed


Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca(2+) from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca(2+) imaging with Fura-2. In a Ca(2+)-free medium, 10-, 60-, and 300-ns stimuli evoked Ca(2+) transients by mobilization of Ca(2+) from the endoplasmic reticulum. With 2mM external Ca(2+), the transients included both extra- and intracellular components. The recruitment of intracellular Ca(2+) increased as the stimulus duration decreased. At the threshold of 200-300nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca(2+) signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca(2+) can be controlled by the duration of the stimulus.

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    • "Caution was exercised not to assume that nsPEFs mimicked plasma membrane electroporation by permeabilizing intracellular membranes. Effects on these vesicles [2] and release of Ca 2+ from intracellular stores [3] [4] [5] [6] [7] or α-granules [8] could be readily explained by permeabilization or electroporation events. However, for the most part, these are assumptions that extend from conventional electroporation effects on plasma membranes. "
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    ABSTRACT: Most, if not all, effects of intense, pulsed electric fields are analyzed in terms of electrical charging of plasma membranes and/or subcellular membranes. However, not all cell responses from nanosecond pulsed electric fields (nsPEFs) are fully explained by poration of cell membranes. Observations that nsPEFs induce a Ca2-dependent dissipation of the mitochondria membrane potential (ΔΨm), which is enhanced when high frequency components are present in fast rise-fall waveforms, is not compatible with a poration event. Ca2 + is shown to have little or no effect on propidium iodide uptake as a measure of plasma membrane poration and consequently intracellular membranes. Since most if not all Ca2 + -regulated events are mediated by proteins, actions of nsPEFs on a protein(s) that regulate and/or affect the mitochondria membrane potential are possible. To show that nsPEFs can directly affect proteins, nsPEFs non-thermally inactivated the catalytic (phosphotransferase) activity of the catalytic subunit of the cAMP-dependent protein kinase, which is the prototype of the protein kinase superfamily that share a common catalytic mechanism and whose functions are highly dependent on their structure. These studies present indirect and direct evidence that nsPEFs can affect proteins and their functions, at least in part, by affecting their structure.
    Full-text · Article · Aug 2014 · Bioelectrochemistry
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    • "These findings were corroborated using both patch clamp and fluorescence microscopy, leading to the conclusion that nsPEF caused nanoporation (formation of nanometer diameter pores) in the plasma membrane [12] [13]. Recent publications have also shown that this " nanopermeabilization, " by allowing the rapid uptake of calcium, activates intracellular signaling pathways and calcium-induced-calcium uptake [14] [15] [16]. The activation of these processes and elevated intracellular calcium concentration can in itself lead to intrinsic apoptosis, mitochondrial depolarization and damage, making the original observations of field-induced intracellular effects of nsPEF suspect. "
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    ABSTRACT: The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular organelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activity of Jurkat and U937 cells after nsPEF. The XF Analyzer uses a transient micro-chamber of only a few μL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in both U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects.
    Full-text · Conference Paper · Feb 2014
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    ABSTRACT: Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, Propidium Iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (15min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.
    Full-text · Article · Dec 2013 · Biochemical and Biophysical Research Communications
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