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.
