Primary pathways of intracellular Ca2+ mobilization by nanosecond pulsed electric field

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca2+ concentration ([Ca2+](i)), which determines multifarious downstream effects. By using fast ratiometric Ca2+ imaging with Fura-2, we quantified the external Ca2+ uptake, compared it with Ca2+ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca2+ channels, so that the nsPEF-induced [Ca2+](i) changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+](i) increase by Ca2+ influx from the outside and Ca2+ efflux from the ER, with the E-field thresholds of about 9 and 19 kV/cm, respectively. Above these thresholds, the amplitude of [Ca2+](i) response increased linearly by 8-10 nM per 1 kV/cm until a critical level between 200 and 300 nM of [Ca2+](i) was reached. If the critical level was reached, the nsPEF-induced Ca2+ signal was amplified up to 3000 nM by engaging the physiological mechanism of Ca2+-induced Ca2+-release (CICR). The amplification was prevented by depleting Ca2+ from the ER store with 100 nM thapsigargin, as well as by blocking the ER inositol-1,4,5-trisphosphate receptors (IP3R) with 50 mu M of 2-aminoethoxydiphenyl borate (2-APB). Mobilization of [Ca2+](i); by nsPEF mimicked native Ca2+ signaling, but without preceding activation of plasma membrane receptors or channels. NsPEF stimulation may serve as a unique method to mobilize [Ca2+](i) and activate downstream cascades while bypassing the plasma membrane receptors. (C) 2012 Elsevier B.V. All rights reserved..