Document Type

Article

Publication Date

2014

Publication Title

Biochimica Et Biophysica Acta: Biomembranes

Volume

1838

Issue

10

Pages

2547-2554

DOI

10.1016/j.bbamem.2014.06.015

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 Ca2+ 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 Ca2+ level from the nominal 2–5 μM to 2 mM for the first 60–90 min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca2+, 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 2 mM Ca2+ in the medium. With the nominal Ca2+, most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2 mM Ca2+, 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-Ca2+-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.

Comments

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Original Publication Citation

Pakhomova, O. N., Gregory, B., Semenov, I., & Pakhomov, A. G. (2014). Calcium-mediated pore expansion and cell death following nanoelectroporation. Biochimica Et Biophysica Acta: Biomembranes, 1838(10), 2547-2554. doi:10.1016/j.bbamem.2014.06.015

ORCID

0000-0003-4950-4130 (Pakhomova), 0000-0002-0302-1355 (Semenov)

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