A large number of intracellular biological responses are closely associated with events that originate at the cell membrane. Often, primary events at or near the membrane (such as externally applied electric fields, surface binding, ion movements etc.) are accompanied with significant alternations of the membrane electric potential. One such consequence (due to E-fields) is the dielectric breakdown of the membrane which results in membrane pore formation. However, despite the application of an homogeneous field, the susceptibility of membranes to dielectric breakdown is found to be dependent not only on the magnitude but also the direction of the electric field as evidenced by the location of the permeabilization site with respect to the electrodes. These observations indicated that a resting membrane potential may be involved. A number of our experimental findings were consistent with this hypothesis. Namely, the permeabilization site on the membrane is reversed from the anode facing hemisphere to that facing the cathode in: (a) high ionic strength medium, (b) the presence of positively charged lipophilic ions, and (c) the presence of indicator molecules with increasing positive charge. On the other hand, depolarization of the bulk-to-bulk membrane potential with the valinomycin had no effect except at [KCl] >150mM. Histamine induced Ca(II) oscillations in HeLa cells has been investigated. To facilitate these in vivo studies, the electropermeabilization apparatus has been improved to include Calcium(II) signal recordings and computer programs for analysis have been developed. It was found neither protein kinase C nor cyclic-AMP dependent protein kinase are involved in the regulation of calcium oscillation in HeLa cells. On the other hand, CaM Kinase II may be involved probably through the activation of Ca-ATPases of the ER. The possibility that it may also be involved in the regulation of inositol triphosphate receptor is being investigated. Introduction of antibodies into the cytoplasm of Acanthamoeba Castellanii by electroporation has been investigated. Preliminary experiments show that fluorescence labeled antibody (FITC-labeled IgG) is successfully incorporated into the cytoplasm.
