Nanosecond pulsed electric field induced dose dependent phosphatidylinositol-(4,5)-bisphosphate signaling and intracellular electro-sensitization

摘要

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol-(4,5)-bisphosphate (PIP2) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose -dependent PIP2 hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP2 hydrolysis: PLC delta-PH-EGFP and GFP-C1-PKC gamma-C-1a. The 50% maximum response occurs with a single 600 ns pulse achieving an effective dose (ED50) of EF similar to 8 kV/cm within our model cell system. At 16.2 kV/cm, the ED50 for the pulse width was 484 ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60 ns 162 kV/cm pulses produced an effect similar to a single 600 ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP2 depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions. (C) 2017 Elsevier B.V. All rights reserved.