EFFECT OF ELECTRIC FIELDS ON BIOMEMBRANES; CELL SIGNALING

在完整细胞上施加足够强的电脉冲 导致瞬时（和可逆）膜孔的产生 不可渗透的分子可以通过其传输。 了解 这一过程的机械基础，核酸的流入 检查了由于电穿孔引起的指示剂染料。 的传播 胞质溶胶中的染料前沿显示出双相时间过程。 快速 流入发生在脉冲之后的前200毫秒内。 较慢的 流入遵循秒的时间尺度，其速率也被发现， 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外地援助， 运输（如电泳、电漂移、电渗等） 由于电脉冲的持续时间（约200微秒）非常长 短于观测到的初始流入的时间尺度（约200 毫秒）。 在本研究的毫秒时间尺度内， 主要由扩散驱动。 因此，突然转向较慢的速度， 染料的上染率是不期望的。 我们认为观察到的断裂 传输速率可能是显著降低（在 数量或尺寸）和/或一些膜孔的完全重新密封 在脉冲后的前几百毫秒结束时。 过氧化氢（H2 O2）在EGF诱导的细胞凋亡中的作用 信号通路进行了研究。 EGF刺激A431细胞 导致细胞内H2 O2浓度短暂升高 但通过将过氧化氢酶掺入细胞中， 电穿孔 电穿孔过氧化氢酶的量约为5 是内源酶的两倍。 过氧化氢酶对过氧化氢的去除作用 还抑制EGF诱导的各种酪氨酸磷酸化， 细胞蛋白质 此外，EGF不能诱导H2 O2的形成。 具有激酶失活EGF受体但正常H2 O2产生的细胞， 在突变受体的细胞中观察到， 自磷酸化位点。 这些结果表明， H2 O2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.