Transport of a divalent cation [Ca(II)] and three DNA indicators [Ethidium Bromide (EB), Propidium Iodide (PI), and Ethidium Homodimer (EthD-1)] across electroporated membranes of several mammalian cell lines and L. pictus sea urchin eggs was found to be selective and asymmetrical. For mammalian cell lines (in low salt medium), Ca(II) and EB preferentially transported across the anode facing cell membrane, while PI and EthD-1 predominantly entered at the site facing the cathode. In high salt medium, the entry site for Ca(II) and EB was reversed to the cathode facing hemisphere, while it remained unchanged for PI and EthD-1. In contrast, the site of entry on sea urchin eggs occurred predominantly at the cathode facing membrane, irrespective of the probe molecule used or the medium ionic strength. In all of these experiments, the observed transport patterns remained unaffected whether the dyes (or ion) were present during or added after the electroporating pulse. Together, the data suggest asymmetric pores are created on both sides of the membrane facing the electrodes with smaller pore size, but higher in number on the anode side and larger pores and lower population on the cathode side. Furthermore, the rate of resealing of the membrane pores is significantly enhanced in high ionic strength medium thus affecting the entry site. The asymmetric transport pattern is neither caused by electrophoresis nor is it due to one-sided membrane breakdown as previously believed. Histamine induced Calcium(II) oscillations in HeLa cells has been investigated. The role of reversible phosphorylation in these oscillations was examined using various activators and inhibitors of protein kinases and phosphatases. Of the kinases examined, only CaMK II was essential. Protein phosphatase inhibitors either abolished ongoing oscillations or prolonged inter-spike intervals. Experiments using P(32)-labeled cells showed the first evidence for in vivo phosphorylation of IP(3) receptor by CaMK II during oscillation. These results show the controlling factors for sustained Ca(II) oscillation in HeLa cells may be repetitive phosphorylation-dephosphorylation cycles involving CaMK II and a type 1 or 2A phosphatase.
