RESUMO
High-amplitude, MV/m, nanosecond pulsed electric fields (nsPEF) have been hypothesized to cause nanoporation of the plasma membrane. Phosphatidylserine (PS) externalization has been observed on the outer leaflet of the membrane shortly after nsPEF exposure, suggesting local structural changes in the membrane. In this study, we utilized fluorescently-tagged Annexin V to observe the externalization of PS on the plasma membrane of isolated Chinese Hamster Ovary (CHO) cells following exposure to nsPEF. A series of experiments were performed to determine the dosimetric trends of PS expression caused by nsPEF as a function of pulse duration, τ, delivered field strength, ED, and pulse number, n. To accurately estimate dose thresholds for cellular response, data were reduced to a set of binary responses and ED50s were estimated using Probit analysis. Probit analysis results revealed that PS externalization followed the non-linear trend of (τ*ED (2))(-1) for high amplitudes, but failed to predict low amplitude responses. A second set of experiments was performed to determine the nsPEF parameters necessary to cause observable calcium uptake, using cells preloaded with calcium green (CaGr), and membrane permeability, using FM1-43 dye. Calcium influx and FM1-43 uptake were found to always be observed at lower nsPEF exposure parameters compared to PS externalization. These findings suggest that multiple, higher amplitude and longer pulse exposures may generate pores of larger diameter enabling lateral diffusion of PS; whereas, smaller pores induced by fewer, lower amplitude and short pulse width exposures may only allow extracellular calcium and FM1-43 uptake.
