title: Calcium-mediated pore expansion and cell death following nanoelectroporation.
abstract: Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca(2+) after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca(2+) level from the nominal 2-5μM to 2mM for the first 60-90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca(2+), the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca(2+) in the medium. With the nominal Ca(2+), most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca(2+), the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca(2+)-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.
authors: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. Electronic address: opakhomo@odu.edu.
authors: Olga N
authors: ON
authors: Pakhomova
authors: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors: Betsy
authors: B
authors: Gregory
authors: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors: Iurii
authors: I
authors: Semenov
authors: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors: Andrei G
authors: AG
authors: Pakhomov
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D053179:Caspase 7 / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D018274:Electroporation; D006801:Humans; D009336:Necrosis / Q000378:metabolism; D009997:Osmotic Pressure; D020298:U937 Cells
chemical_list: C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D002118:Calcium
keywords: Apoptosis; Calcium; Electropermeabilization; Electroporation; Nanosecond pulse; Necrosis