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

Abstract

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. 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 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 3,000 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 μM 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 activate [Ca2+]i and downstream cascades while bypassing the plasma membrane receptors.

Investigation of a direct effect of nanosecond pulse electric fields on mitochondria

“…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.…”

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity

“…Except for using both the bipolar and monopolar pulse generators, the exposure procedures were the same as described previously [5, 6]. Pulses were delivered to selected cells on a coverslip with a pair of 0.1-mm-diameter tungsten rods.…”

“…The downstream effects of nsEP range from cell uptake of membrane-impermeable solutes [8, 9] and transient calcium mobilization [5, 6, 10, 11] to destruction of the cytoskeleton [12], swelling and blebbing [7], and necrotic or apoptotic cell death [1, 2, 13-15]. The membrane defects produced by nsEP have been intensively studied experimentally [7, 16, 17] as well as by molecular dynamics [18-21] and other simulation techniques [22-26], but their exact nature remains elusive.…”

“…The nanopores are remarkably stable, with a lifetime on the order of minutes [8, 13, 16, 17, 27] and show complex conductive properties akin to native ion channels, such as voltage sensitivity, current rectification, and ion selectivity [16, 17]. Mild nsEP stimulation was proposed as a unique method of cell activation that can bypass plasma membrane receptors and channels [5, 6, 10, 28]. …”

Dose dependent translocations of fluorescent probes of PIP2hydrolysis in cells exposed to nanosecond pulsed electric fields

“…The exposure of cells to nanosecond pulsed electric fields (nsPEF) produces multifarious effects, including nuclear granulation, intracellular calcium bursts, cytoskeletal changes, stimulation of action potentials, blebbing and swelling, and initiation of apoptotic cell death [1][2][3][4][5][6][7][8][9]. Most of these processes require ion channels or membrane transport proteins to be present in the cellular membrane.…”