Calcium bursts induced by nanosecond electric pulses.

We report here real-time imaging of calcium bursts in human lymphocytes exposed to nanosecond, megavolt-per-meter pulsed electric fields. Ultra-short (less than 30 ns), high-field (greater than 1 MV/m), electric pulses induce increases in cytosolic calcium concentration and translocation of phosphatidylserine (PS) to the outer layer of the plasma membrane in Jurkat T lymphoblasts. Pulse-induced calcium bursts occur within milliseconds and PS externalization within minutes. Caspase activation and other indicators of apoptosis follow these initial symptoms of nanosecond pulse exposure. Pulse-induced PS translocation is observed even in the presence of caspase inhibitors. Ultra-short, high-field, electroperturbative pulse effects differ substantially from those associated with electroporation, where pulses of a few tens of kilovolts-per-meter lasting a few tens of microseconds open pores in the cytoplasmic membrane. Nanosecond pulsed electric fields, because their duration is less than the plasma membrane charging time, develop voltages across intracellular structures without porating the cell.

[1]  R. Rizzuto,et al.  Mitochondrial Ca2+ Uptake Requires Sustained Ca2+ Release from the Endoplasmic Reticulum* , 2003, The Journal of Biological Chemistry.

[2]  M. Berridge,et al.  Elementary [Ca2+]i signals generated by electroporation functionally mimic those evoked by hormonal stimulation , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  P. Bernardi.  Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization. , 1992, The Journal of biological chemistry.

[4]  H. Schwan,et al.  On the possibility of nonthermal biological effects of pulsed electromagnetic radiation. , 1970, Biophysical journal.

[5]  E. Dumay,et al.  Effects of pulsed electric fields on ovalbumin solutions and dialyzed egg white. , 2000, Journal of agricultural and food chemistry.

[6]  J. Imboden,et al.  Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores , 1985, The Journal of experimental medicine.

[7]  L. Mir,et al.  Therapeutic perspectives of in vivo cell electropermeabilization. , 2001, Bioelectrochemistry.

[8]  N. Mohandas,et al.  Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  K. Schoenbach,et al.  Nanosecond, high‐intensity pulsed electric fields induce apoptosis in human cells , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  K. Schoenbach,et al.  Self-consistent simulations of electroporation dynamics in biological cells subjected to ultrashort electrical pulses. , 2001, Physical review. E, Statistical, nonlinear, and soft matter physics.

[11]  C. Haest,et al.  Transbilayer reorientation of phospholipid probes in the human erythrocyte membrane. Lessons from studies on electroporated and resealed cells. , 1997, Biochimica et biophysica acta.

[12]  U. Zimmermann,et al.  Electric Field Pulses Can Induce Apoptosis , 1999, The Journal of Membrane Biology.

[13]  K. Schoenbach,et al.  The effects of intense submicrosecond electrical pulses on cells. , 2003, Biophysical journal.

[14]  Kenneth R. Foster,et al.  Thermal and nonthermal mechanisms of interaction of radio-frequency energy with biological systems , 2000 .

[15]  F Sachs,et al.  Voltage-induced membrane movement , 2001, Nature.

[16]  P. Cullen,et al.  Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  L. Cantley,et al.  Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx , 1985, Cell.

[18]  K. Schoenbach,et al.  Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition , 2001, PPPS-2001 Pulsed Power Plasma Science 2001. 28th IEEE International Conference on Plasma Science and 13th IEEE International Pulsed Power Conference. Digest of Papers (Cat. No.01CH37251).

[19]  J. Roth,et al.  Bax-mediated Ca2+ Mobilization Promotes Cytochromec Release during Apoptosis* , 2002, The Journal of Biological Chemistry.

[20]  P. Williamson,et al.  Ca2+ induces transbilayer redistribution of all major phospholipids in human erythrocytes. , 1992, Biochemistry.

[21]  W. Chen,et al.  Electric field-induced functional reductions in the K+ channels mainly resulted from supramembrane potential-mediated electroconformational changes. , 1998, Biophysical journal.

[22]  A. Zweifach,et al.  Evidence that cytosolic calcium increases are not sufficient to stimulate phospholipid scrambling in human T-lymphocytes. , 2002, The Biochemical journal.

[23]  M. Madesh,et al.  Calcium signaling and apoptosis. , 2003, Biochemical and biophysical research communications.

[24]  L. Chernomordik,et al.  Voltage-induced nonconductive pre-pores and metastable single pores in unmodified planar lipid bilayer. , 2001, Biophysical journal.

[25]  M. Sanderson,et al.  A role for Ca(2+)-conducting ion channels in mechanically-induced signal transduction of airway epithelial cells. , 1994, Journal of cell science.

[26]  J. García-Sancho,et al.  Mitochondrial Ca2+-induced Ca2+ Release Mediated by the Ca2+ Uniporter , 2001 .

[27]  A. Driessen,et al.  The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid , 1998, Journal of bacteriology.

[28]  J Teissié,et al.  Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. , 1990, Biophysical journal.

[29]  W. Krassowska,et al.  Modeling electroporation in a single cell. I. Effects Of field strength and rest potential. , 1999, Biophysical journal.

[30]  S. Boxer,et al.  Electric field-induced critical demixing in lipid bilayer membranes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  K. Schoenbach,et al.  Intracellular effect of ultrashort electrical pulses , 2001, Bioelectromagnetics.

[32]  R. Penner,et al.  The Store-Operated Calcium Current ICRAC: Nonlinear Activation by InsP3 and Dissociation from Calcium Release , 1997, Cell.

[33]  T. Pozzan,et al.  Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. , 1993, Science.

[34]  P. Sims,et al.  Isolation of an Erythrocyte Membrane Protein that Mediates Ca2+-dependent Transbilayer Movement of Phospholipid* , 1996, The Journal of Biological Chemistry.

[35]  Sten Orrenius,et al.  Calcium: Regulation of cell death: the calcium–apoptosis link , 2003, Nature Reviews Molecular Cell Biology.

[36]  P. Fellmann,et al.  Ultrafast Glycerophospholipid-selective Transbilayer Motion Mediated by a Protein in the Endoplasmic Reticulum Membrane (*) , 1996, The Journal of Biological Chemistry.

[37]  C. Kunzelmann-Marche,et al.  Regulation of Phosphatidylserine Transbilayer Redistribution by Store-operated Ca2+ Entry , 2001, The Journal of Biological Chemistry.

[38]  F. Sachs,et al.  Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. , 1989, Science.

[39]  Laura Marcu,et al.  Pulse generators for pulsed electric field exposure of biological cells and tissues , 2003 .

[40]  J. Weaver,et al.  Theory of electroporation of planar bilayer membranes: predictions of the aqueous area, change in capacitance, and pore-pore separation. , 1994, Biophysical journal.

[41]  V. Fadok,et al.  Loss of Phospholipid Asymmetry and Surface Exposure of Phosphatidylserine Is Required for Phagocytosis of Apoptotic Cells by Macrophages and Fibroblasts* , 2001, The Journal of Biological Chemistry.

[42]  H. Itoh,et al.  Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential. , 1993, Biophysical journal.

[43]  Laura Marcu,et al.  Ultrashort pulsed electric fields induce membrane phospholipid translocation and caspase activation: differential sensitivities of Jurkat T lymphoblasts and rat glioma C6 cells , 2003 .

[44]  J. Nagelkerke,et al.  Regional Loss of the Mitochondrial Membrane Potential in the Hepatocyte Is Rapidly Followed by Externalization of Phosphatidylserines at That Specific Site during Apoptosis* , 2003, The Journal of Biological Chemistry.

[45]  J Teissié,et al.  Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane. , 1997, Biophysical journal.

[46]  A. Schroit,et al.  Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. , 1997, Blood.

[47]  Y. Feldman,et al.  Time domain dielectric spectroscopy study of human cells. II. Normal and malignant white blood cells. , 1999, Biochimica et biophysica acta.

[48]  Robert Plonsey,et al.  Electrical stimulation of excitable cells-a model approach , 1988, Proc. IEEE.

[49]  S. Orrenius,et al.  The role of calcium in the regulation of apoptosis. , 1996, Journal of leukocyte biology.

[50]  J Teissié,et al.  Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse. , 1999, Biophysical journal.

[51]  G. Kroemer,et al.  Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death , 1995, The Journal of experimental medicine.

[52]  L. Genestier,et al.  Caspase-Independent Phosphatidylserine Exposure During Apoptosis of Primary T Lymphocytes1 , 2002, The Journal of Immunology.

[53]  D. Green,et al.  Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl , 1995, The Journal of experimental medicine.

[54]  S. Bunnell,et al.  T Cell Receptor–initiated Calcium Release Is Uncoupled from Capacitative Calcium Entry in Itk-deficient T Cells , 1998, The Journal of experimental medicine.

[55]  T. Mcdonald,et al.  Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. , 1994, Physiological reviews.

[56]  Ulrich Zimmermann,et al.  Dielectric Breakdown of Cell Membranes , 1974 .

[57]  Ulrich Zimmermann,et al.  Electromanipulation of cells , 1996 .

[58]  M. Hoth,et al.  Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes , 1997, The Journal of cell biology.

[59]  J. Breittmayer,et al.  Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells. , 1996, The Biochemical journal.

[60]  A. Schroit,et al.  Immune Clearance of Phosphatidylserine-expressing Cells by Phagocytes , 1997, The Journal of Biological Chemistry.

[61]  V. Gogvadze,et al.  Fas‐triggered phosphatidylserine exposure is modulated by intracellular ATP , 2002, FEBS letters.

[62]  I. Sugár.  A theory of the electric field-induced phase transition of phospholipid bilayers. , 1979, Biochimica et biophysica acta.

[63]  F. Sachs,et al.  Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells , 2000, The Journal of Membrane Biology.

[64]  P. Williamson,et al.  Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes , 1995, The Journal of experimental medicine.