title: 600 ns pulse electric field-induced phosphatidylinositol4,5-bisphosphate depletion.
abstract: The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.
authors: National Research Council, Oak Ridge Institute for Science and Education, JBSA Fort Sam Houston, TX 78234, USA; General Dynamics Information Technology, Fort Sam Houston, San Antonio, TX, USA. Electronic address: gtolstykh@gmail.com.
authors: Gleb P
authors: GP
authors: Tolstykh
authors: Air Force Research Laboratory, 711th Human Performance Wing, Human Effectiveness Directorate, Bioeffects Division, JBSA Fort Sam Houston, TX 78234, USA.
authors: Hope T
authors: HT
authors: Beier
authors: Department of Radiology, University of Texas Health Science Center San Antonio, TX 78229, USA.
authors: Caleb C
authors: CC
authors: Roth
authors: National Research Council, Oak Ridge Institute for Science and Education, JBSA Fort Sam Houston, TX 78234, USA.
authors: Gary L
authors: GL
authors: Thompson
authors: Air Force Research Laboratory, 711th Human Performance Wing, Human Effectiveness Directorate, Bioeffects Division, JBSA Fort Sam Houston, TX 78234, USA.
authors: Bennett L
authors: BL
authors: Ibey
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004075:Diglycerides / Q000378:metabolism; D004560:Electricity; D018274:Electroporation / Q000379:methods*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006868:Hydrolysis; D042541:Intracellular Space / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000172:deficiency* / Q000378:metabolism; D013997:Time Factors
chemical_list: D004075:Diglycerides; D018129:Phosphatidylinositol Phosphates; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium
keywords: Calcium; Electric pulse; Nanopore; PIP(2) depletion; Phospholipase C