Phagocytic clearance of electric field induced 'apoptosis-mimetic' cells.

Cells undergoing apoptosis lose lipid asymmetry that is often manifested by the exposure of phosphatidylserine (PS) to the outer surface of the cell membrane. Macrophages and other cell types recognize externalized PS to signal phagocytosis, thereby eliciting a non-inflammatory response. PS exposure is obligatory in the recognition and clearance of apoptotic cells. Here, we find that externally applied moderate electric field induces PS externalization in a mouse B-cell (FOX-NY) membrane without procaspase-3 activation, a major characteristic of apoptotic cells. The field-induced PS inversion is caused as a result of electroporation and/or a process involving membrane reorganizations and recovery that ensues following field exposure. Using a mouse macrophage cell line (J7444A.1) from the same strain, we show phagocytic clearance of PS expressing B-cells and demonstrate that this is in part due to the apoptosis mimicry of the field exposed cells.

Reversible glutathionylation regulates actin polymerization in A431 cells.

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization. In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 microM comparable with its IC(50) in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation.

Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles.

An externally applied electric field across vesicles leads to transient perforation of the membrane. The distribution and lifetime of these pores was examined using 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid vesicles using a standard fluorescent microscope. The vesicle membrane was stained with a fluorescent membrane dye, and upon field application, a single membrane pore as large as approximately 7 microm 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 formation of many small pores is implicated by the data. Analysis of pre- and post-field fluorescent vesicle images, as well as images from negatively stained electron micrographs, indicate that pore formation is associated with a partial loss of the phospholipid bilayer from the vesicle membrane. Up to approximately 14% of the membrane surface could be lost due to pore formation. Interestingly, despite a clear difference in the size distribution of the pores observed, the effective porous areas at both hemispheres was approximately equal. Ca(2+) influx measurements into perforated vesicles further showed that pores are essentially resealed within approximately 165 ms after the pulse. The pore distribution found in this study is in line with an earlier hypothesis (E. Tekle, R. D. Astumian, and P. B. Chock, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11512--11516) of asymmetric pore distribution based on selective transport of various fluorescent markers across electroporated membranes.

Inositol tetrakisphosphate as a frequency regulator in calcium oscillations in HeLa cells.

Cellular signaling mediated by inositol (1,4,5)trisphosphate (Ins(1, 4,5)P(3)) results in oscillatory intracellular calcium (Ca(2+)) release. Because the amplitude of the Ca(2+) spikes is relatively invariant, the extent of the agonist-mediated effects must reside in their ability to regulate the oscillating frequency. Using electroporation techniques, we show that Ins(1,4,5)P(3), Ins(1,3,4, 5)P(4), and Ins(1,3,4,6)P(4) cause a rapid intracellular Ca(2+) release in resting HeLa cells and a transient increase in the frequency of ongoing Ca(2+) oscillations stimulated by histamine. Two poorly metabolizable analogs of Ins(1,4,5)P(3), Ins(2,4,5)P(3), and 2,3-dideoxy-Ins(1,4,5)P(3), gave a single Ca(2+) spike and failed to alter the frequency of ongoing oscillations. Complete inhibition of Ins(1,4,5)P(3) 3-kinase (IP3K) by either adriamycin or its specific antibody blocked Ca(2+) oscillations. Partial inhibition of IP3K causes a significant reduction in frequency. Taken together, our results indicate that Ins(1,3,4,5)P(4) is the frequency regulator in vivo, and IP3K, which phosphorylates Ins(1,4, 5)P(3) to Ins(1,3,4,5)P(4), plays a major regulatory role in intracellular Ca(2+) oscillations.

Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation.

Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers in receptor signaling pathways. The possible role of ROS in epidermal growth factor (EGF) signaling was therefore investigated. Stimulation of A431 human epidermoid carcinoma cells with EGF resulted in a transient increase in the intracellular concentration of ROS, measured with the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by incorporation of catalase into the cells by electroporation. The elimination of H2O2 by catalase also inhibited the EGF-induced tyrosine phosphorylation of various cellular proteins including the EGF receptor and phospholipase C-gamma1. The dependence of H2O2 production on the intrinsic tyrosine kinase activity of the EGF receptor and the autophosphorylation sites located in its COOH-terminal tail was investigated. EGF failed to induce H2O2 generation in cells expressing a kinase-inactive EGF receptor. However, normal H2O2 generation was observed in cells expressing a mutant receptor from which the 126 COOH-terminal amino acids had been deleted to remove four (out of the total of five) autophosphorylation sites. These results suggest that EGF-induced H2O2 formation requires the kinase activity but probably not the autophosphorylation sites of the EGF receptor and that inhibition of protein tyrosine phosphatase activity by H2O2 may be required for EGF-induced protein tyrosine phosphorylation to be manifested.

Reversible phosphorylation as a controlling factor for sustaining calcium oscillations in HeLa cells: Involvement of calmodulin-dependent kinase II and a calyculin A-inhibitable phosphatase.

The role of reversible phosphorylation in histamine-induced Ca2+ oscillations in HeLa cells has been investigated by using various activators and inhibitors of protein kinases and phosphatases. Electroporation was employed to introduce impermeable materials into single cells, which proved to be a useful and convenient tool. Of the kinases examined, cAMP-dependent kinase, protein kinase C, and calmodulin-dependent kinase II (CaMK II), only CaMK II was essential. When added during oscillations, both W-7, a calmodulin antagonist, and KN-62, a specific CaMK II inhibitor, caused one large Ca2+ spike before halting the process. Introduction of the Ca2+/calmodulin-independent catalytic domain of CaMK II into the cells forestalled their response to histamine. These results show that intracellular Ca2+ cannot oscillate when CaMK II is locked in either the inactive or the stimulated state. External Ca2+ electroporated into cells preloaded with the catalytic domains was quickly removed (but not when the cells were pretreated with the endoplasmic reticulum Ca(2+)-ATPase inhibitor, tapsigargin), indicating that the ATP-driven Ca2+ pump was somehow activated by CaMK II. Protein phosphatase inhibitors calyculin A and okadaic acid abolished ongoing oscillations and, when added at low concentrations, prolonged the interspike interval. Immunoprecipitation experiments with 32P(i)-labeled cells provided the first evidence that inositol 1,4,5-trisphosphate receptor (IP3R) was phosphorylated by CaMK II in vivo. The extent of phosphorylation was increased in the presence of histamine, significantly enhanced by calyculin A, and greatly reduced by W-7. Our observations are consistent with the concept that repetitive phosphorylation-dephosphorylation cycles regulating IP3R and Ca2+ pumps are a controlling factor for sustained Ca2+ oscillations in HeLa, and possibly other, cells.

Selective and asymmetric molecular transport across electroporated cell membranes.

Transport of a divalent cation (Ca2+) and three DNA indicators [ethidium bromide (EB), propidium iodide (PI), and ethidium homodimer (EthD-1)] across electroporated membranes of several mammalian cell lines was found to be selective and asymmetrical. In low salt medium, Ca2+ and EB were preferentially transported across the anodefacing cell membrane while PI and EthD-1 predominately entered at the site facing the cathode. In high salt medium, the entry site for Ca2+ and EB was reversed to the cathode-facing hemisphere while it remained unchanged for PI and EthD-1. In all these experiments, the observed transport patterns remained unaffected whether the dyes (or ion) were present during or added after the electroporating pulse. The data suggest that asymmetric pores are created on both sides of the membrane facing the electrodes, with smaller pore size (but greater in number) on the anode side and larger pores (with a 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 induced by the externally applied electric field nor due to one-sided membrane breakdown as previously believed.

Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3T3 cells.

Using the plasmid DNA pSV2-neo (which, when integrated into the cellular genome confers resistance to the antibiotic G418 for selection), we examined and compared the transfection efficiency on NIH 3T3 cells electropermeabilized by applying a sequence of high-frequency unipolar or bipolar square waves or a single square pulse. Results show that a bipolar square wave is, at least, 1.7- and 5.5-fold more efficient than the unipolar square wave and single square pulse, respectively. In the range of electric field strength used for optimum transfection, the survivability of electropermeabilized cells was comparable between the unipolar and bipolar square waves but fell considerably with the single square pulse. Qualitative comparison of cell permeabilization induced by the three types of wave forms and monitored by ethidium bromide uptake revealed that only the bipolar square wave permeabilizes the cell membrane symmetrically at the two hemispheres facing the electrodes. With unipolar square wave or single square pulse, the membrane is permeabilized either on one side or asymmetrically. Taken together, our result suggests that permeabilization of the membrane at multiple sites without affecting cell survivability may account for the improvements in transfection efficiency observed with bipolar oscillating electric fields.

Electro-permeabilization of cell membranes: effect of the resting membrane potential.

Electric field induced permeabilization of cell membranes is an important technique for gene transfection and cell hybridization. Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed. In light of these controversial observations, we conducted a systematic study of electroporation of NIH3T3 cells with varying electric field strength, waveform and frequency. Our data revealed that (i) symmetrical permeabilization of the cell membrane occurs only with bipolar a.c. fields. (ii) When a critical membrane breakdown potential, Vc, is applied using either an unipolar a.c. fields or a single d.c. square pulse, the cell membrane becomes permeabilized only at the hemisphere facing the positive electrode. (iii) When the pulse-induced membrane potential, Vm, is approximately equal to or larger than the intrinsic membrane potential (i.e. using d.c. or unipolar a.c. field), asymmetric permeabilization was observed with the hemisphere facing the positive electrode being most permeable. (iv) The rate of fluorescent indicator uptake is dependent on the concentration of the indicator. These results indicate that electro-permeabilization of cell membranes is affected by its resting potential and that electroosmosis is not the dominant mechanism for the cellular uptake of foreign molecules in electroporation.