title: Primary pathways of intracellular Ca(2+) mobilization by nanosecond pulsed electric field.
abstract: Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which determines multifarious downstream effects. By using fast ratiometric Ca(2+) imaging with Fura-2, we quantified the external Ca(2+) uptake, compared it with Ca(2+) release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca(2+) channels, so that the nsPEF-induced [Ca(2+)](i) changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca(2+)](i) increase by Ca(2+) influx from the outside and Ca(2+) efflux from the ER, with the E-field thresholds of about 9 and 19kV/cm, respectively. Above these thresholds, the amplitude of [Ca(2+)](i) response increased linearly by 8-10nM per 1kV/cm until a critical level between 200 and 300nM of [Ca(2+)](i) was reached. If the critical level was reached, the nsPEF-induced Ca(2+) signal was amplified up to 3000nM by engaging the physiological mechanism of Ca(2+)-induced Ca(2+)-release (CICR). The amplification was prevented by depleting Ca(2+) from the ER store with 100nM thapsigargin, as well as by blocking the ER inositol-1,4,5-trisphosphate receptors (IP(3)R) with 50μM of 2-aminoethoxydiphenyl borate (2-APB). Mobilization of [Ca(2+)](i) by nsPEF mimicked native Ca(2+) signaling, but without preceding activation of plasma membrane receptors or channels. NsPEF stimulation may serve as a unique method to mobilize [Ca(2+)](i) and activate downstream cascades while bypassing the plasma membrane receptors.
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. Iurii.semenov@gmail.com
authors.forename: Iurii
authors.initials: I
authors.lastname: Semenov
authors.forename: Shu
authors.initials: S
authors.lastname: Xiao
authors.forename: Andrei G
authors.initials: AG
authors.lastname: Pakhomov
mesh_terms: D000818:Animals; D001703:Biophysics / Q000379:methods; D002021:Buffers; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry*; D015220:Calcium Channels / Q000187:drug effects; D020013:Calcium Signaling / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D004563:Electrochemistry / Q000379:methods; D018274:Electroporation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007700:Kinetics; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D002021:Buffers; D015220:Calcium Channels; D019284:Thapsigargin; D002118:Calcium