Tolstykh Gleb P, Beier Hope T, Roth Caleb C, Thompson Gary L, Ibey Bennett L
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.
Air Force Research Laboratory, 711th Human Performance Wing, Human Effectiveness Directorate, Bioeffects Division, JBSA Fort Sam Houston, TX 78234, USA.
Bioelectrochemistry. 2014 Dec;100:80-7. doi: 10.1016/j.bioelechem.2014.01.006. Epub 2014 Jan 29.
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.
纳秒级脉冲电场（nsPEF）诱导的钙离子释放与nsPEF诱导的磷脂酰肌醇 - 4,5 - 二磷酸（PIP2）水解之间的相互作用尚未得到充分理解。为了更好地理解这种相互关系，我们监测了负载钙黄绿素 - 1 AM的细胞内钙离子变化，以及转染了两种荧光报告基因之一的细胞中PIP2水解产物（肌醇 - 1,4,5 - 三磷酸（IP3）和二酰基甘油（DAG））的生成：PLCδ - PH - EGFP或GFP - C1 - PKCγ - C1a。测定了暴露后的荧光差异百分比（ΔF %）。在nsPEF作用下，我们发现，在没有细胞外钙离子的情况下，nsPEF暴露期间释放的IP3数量（ΔF 6%±3，n = 22），与存在钙离子时的反应相比有所减少（ΔF 84%±15，n = 20）。在没有细胞外钙离子的情况下（ΔF 29%±5，n = 25）以及在暴露于毒胡萝卜素的细胞中（ΔF 40%±12，n = 15），DAG的生成与存在细胞外钙离子的细胞（ΔF 22±6%，n = 18）相比没有统计学差异。这一发现表明，细胞内钙离子浓度的变化并非是观察到的反应的唯一驱动因素。有趣的是，在没有钙离子的情况下产生的DAG在面向电极的膜区域附近最强，而细胞外钙离子的存在则导致全细胞反应。所报道的钙离子动力学观察结果与IP3和DAG生成相结合表明，nsPEF可能对质膜内的磷脂产生直接影响。
