EFFECT OF ELECTRIC FIELDS ON BIOMEMBRANES; CELL SIGNALING

在完整的细胞间施加足够强的电脉冲 导致产生瞬变(和可逆)膜孔 不透水的分子可以通过它来运输。要理解 这一过程的机制基础是一种核酸的流入 对电穿孔引起的指示剂染料进行了检测。的传播 胞浆中的染料前沿呈现双相时间过程。一次快速的 内流发生在脉冲后的前200毫秒内。一个速度较慢的 涌入紧随其后的是秒时间尺度，其速度也被发现 与脉冲介质的离子强度成反比。 最初的快速涌入不能归因于现场协助 运输(如电泳法、电漂移法、电渗法等) 因为电脉冲的持续时间(大约200微秒) 短于观察到的初始流入的时间尺度(约200 Msecs)。在本研究的MSECS时间尺度中，运输是 主要是由扩散驱动的。因此，突然转向较慢的速度 染料上染率不会是预期的。我们提出了观察到的突破口 传输速率可能是显著降低的结果(在 数量或大小)和/或完全重新密封一些膜孔 在脉冲后的第一个几百毫秒结束时。 过氧化氢(H_2O_2)在表皮生长因子诱导中的可能作用 对信号传导途径进行了研究。表皮生长因子对A431细胞的刺激作用 导致细胞内过氧化氢浓度一过性增加 但通过将过氧化氢酶掺入细胞内而完全被取消 电穿孔。电穿孔过氧化氢酶的量约为5 是内源酶的三倍。过氧化氢酶对过氧化氢的清除作用 还可抑制EGF诱导的多种酪氨酸磷酸化 细胞蛋白质。此外，EGF不能诱导细胞产生H_2O_2 具有非激活型EGF受体但正常产生过氧化氢的细胞 在缺乏突变受体的细胞中观察到五分之四 自动磷酸化位点。这些结果表明， 过氧化氢作为细胞内的信使。

Application of a sufficiently intense electric pulse across intact cells results in the creation of transient (and reversible) membrane pores through which impermeable molecules can be transported. To understand the mechanistic basis of this processes, the influx of a nucleic acid indicator dye due to electroporation was examined. The propagation of the dye front in the cytosol showed a biphasic time course. A rapid influx occurs within the first 200 msecs after the pulse. A slower influx follows in the seconds time scale, whose rate was also found to be inversely correlated with the ionic strength of the pulsing medium. The initial rapid influx cannot be attributed to a field assisted transport (e.g. electrophoresis, electrical drift, electroosmosis, etc.) as the duration of the electric pulse (about 200 microsecs) is much shorter than the observed time scale of the initial influx (about 200 msecs). In the msecs time scale of this study, the transport is primarily driven by diffusion. Thus, a sudden shift to a slower rate for the dye uptake would not be expected. We propose the observed break in the transport rate may be a consequence of a significant reduction (in number or size) and/or complete resealing of some of the membrane pores by the end of the first few hundred msecs after the pulse. The possible role of hydrogen peroxide (H2O2) in the EGF induced signaling pathway was investigated. Stimulation of A431 cells with EGF resulted in a transient increase in the intracellular H2O2 concentration but was completely abolished by incorporation of catalase into cells by electroporation. The amount of electroporated catalase was about five times that of the endogenous enzyme. The elimination of H2O2 by catalase also inhibited the EGF induced tyrosine phosphorylation of various cellular proteins. Furthermore, EGF failed to induce H2O2 formation in cells with a kinase-inactive EGF receptor but normal H2O2 production was observed in cells with a mutant receptor lacking four out of five autophosphorylation sites. These results suggest a possible role for H2O2 as an intracellular messenger.