The induction and distribution of electric field-induced membrane pores were examined using 1,2-Di-oleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid vesicles (>10 micromolars) and a standard fluorescent microscope. The vesicles were stained with a fluorescent membrane dye. Upon field application, a single membrane pore as large as ~6 micrometers in diameter was observed at the vesicle membrane facing the negative electrode. At the anode-facing hemisphere, large and visible pores are seldom found, but the formation of many small pores is implicated by the data. Image analysis revealed that these pores were created by a partial loss of the phospholipid bilayer from the vesicle membrane. Up to ~14% of the membrane surface could be lost due to pore formation. Despite a clear difference in the size distribution of the pores observed, the effective porous areas at both hemispheres were approximately equal. Based on measurements of Ca(II) influx rates into porated vesicles, we found the field-induced pores were resealed about 164 msecs after the pulse. 2',7'-dichlorofluorescin diacetate (DCF-DA) is an oxidation-sensitive fluorescent dye widely used in the detection of intracellular-generated reactive oxygen species (ROS). These ROS species may include, among others, the superoxide anion radical, hydrogen peroxide, alkoxyl radical, peroxyl radical, lipid peroxides, nitric oxide, peroxynitrite, and hydroxyl radical. The extent, rate, and relative ability of these reactive oxygen species to oxidize DCF is, however, not clear. Using both EPR (Electron Paramagnetic Resonance) and spectroflurometeric methods, a number of in vitro assays are being developed to investigate the relative contribution to DCF oxidation by these cellular oxidants.
