Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.     

Alternating electric fields stimulate ATP synthesis in Escherichia coli

External alternating electric fields of low intensity stimulated membrane bound ATP synthesis in starving Escherichia coli cells with electric field amplitudes of 2.5-50 V/cm and a frequency optimum at 100 Hz. The model of electrocon-formational coupling was used to analyze the frequency and amplitude responses of ATP synthesis. Two relaxation frequencies of the system were obtained at 44 Hz and 220 Hz, and an estimate of roughly 12 was obtained as the effective charge displacement for the catalytic cycle of ATP synthesis.     

Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation

Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.     

Evaporation and plant damage in electric fields

Beans exposed to intense static electric fields are observed to experience large scale damage although corona occurs from only a few points on the leaves. Increased evaporative loss is considered to be the physical mechanism which accounts for the observed effects.     

Microtubule arrays in regeneratingMougeotia protoplasts may be oriented by electric fields

Summary Initially non-polar protoplasts of the green algaMougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 Vcm^–1 (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10 mV/cell), 53% of protoplasts were oriented within +- 10° of the 0/180° axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10 mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two foci that appears within 3 to 4 h, and the subsequent organization of microtubules by 8 to 12 h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.Abbreviations PBS phosphate buffered saline - PMM protoplast maintenance medium - DMM dilute maintenance medium - MES 2(N-morpholino)ethanesulfonic acid - TRIS tris(hydroxymethyl)aminomethane - ANOVA analysis of variance     

The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter.

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.     

Structural transition produced by electric fields in aqueous sodium deoxyribonucleate

It was found that the birefringence of aqueous solutions of sodium DNA is anomalous when electric fields of high intensity (≥10⁴ v/cm) are applied. The magnitude of the birefringence first rose upon application of the orienting pulse, then fell as the field was sustained above a critical value. The occurrence of the effect depended upon macromolecular and electrolyte concentrations. Upon removal of the field, the birefringence was rapidly restored and then it decayed with an increase of the reorientational relaxation times, relative to those observed below the critical field. It is proposed that the electric field may cause aggregation of the macromolecules and then produce a structural transition concomitant with the electric field orientation effect. This transition may correspond to the “B” “A” structures identified in x-ray studies, or to a “B” “V” structure change, where “V” is a postulated new helical form stabilized by cooperative interactions of base and dipoles in the electric field. Field induced transitions of this type would be of interest in connection with molecular mechanisms of transport through membranes, nerve impulse transmission, or information storage.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Oomycete plant pathogens use electric fields to target roots

Plant roots generate electrical currents and associated electrical fields as a consequence of electrogenic ion transport at the root surface. Here we demonstrate that the attraction of swimming zoospores of oomycete plant pathogens to plant roots is mediated in part by electrotaxis in natural root-generated electric fields. The zones of accumulation of anode- or cathode-seeking zoospores adjacent to intact and wounded root surfaces correlated with their in vitro electrotactic behavior. Manipulation of the root electrical field was reflected in changes in the pattern of zoospore accumulation and imposed focal electrical fields were capable of overriding endogenous signals at the root surface. The overall pattern of zoospore accumulation around roots was not affected by the presence of amino acids at concentrations expected within the rhizosphere, although higher concentrations induced encystment and reduced root targeting. The data suggest that electrical signals can augment or override chemical ones in mediating short-range tactic responses of oomycete zoospores at root surfaces.     

Nuclear magnetic resonance studies of sodium/calcium exchange in frog perfused, beating hearts

This study explores the effect of extracellular Ca2+ concentration ([Ca2+]o), on the intracellular Na+ concentration ([Na+]i), in frog intact hearts using nuclear magnetic resonance spectroscopy, which allows for the measurement of [Na+]i in perfused, beating hearts. Decreases in [Ca2+]o yielded marked increases in [Na+]i. A similar effect was seen during inhibition of the Na+/K+ pump and was fully reversible. This sensitivity of [Na+]i to [Ca2+]o, previously observed using microelectrodes, supports a crucial physiological role for Na+/Ca2+ exchange in frog intact, beating hearts.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

Parathyroid hormone increases sodium/calcium exchange activity in renal cells and the blunting of the response in aging

Na+-dependent Ca2+ efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+-dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biologically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells isolated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-dependent Ca2+ efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid-stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin-stimulated Na+-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. These findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. These results may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age.     

Polyethylene glycol and electric field treatment of plant protoplasts: characterization of some membrane properties

Petiolar protoplasts of a dihaploid line of winter oilseed rape Brassica napus L. ssp. oleifera were exposed to fusogenic polyethylene glycol (PEG) and electric field treatments. The surface properties and stability of membrane components of the treated protoplasts were investigated by contact angle measurements in aqueous two-phase systems and differential scanning calorimetry, respectively. The leakage of intracellular components was estimated with respect to amino acids, proteins and DNA. Both fusogenic treatments resulted in the same apparent changes in membrane surface hydrophobicity and the same destabilization of membrane components. However, the PEG-treated protoplasts were more leaky than both the control and the electric field-treated protoplasts. The results indicate that the molecular mechanisms of PEG- and electrical field-induced fusion are similar. However, the effects of the latter appear to be less harmful presumably because the parameters for electric field treatment are more easily controlled.     

Characterization of a calcium/proton antiporter and an electrogenic calcium transporter in membrane vesicles from Azotobacter vinelandii

Ca2+ transport across the membrane of vesicles derived from Azotobacter vinelandii was studied in the absence of respiration or functioning ATPase. Two facilitated diffusion systems were found. One, an electroneutral Ca2+/2H+ antiporter, responded to an artificially imposed deltapH, was heat-labile, and was insensitive to low concentrations of ruthenium red and lanthanides. The second, an electrogenic transporter, responded to an electrical membrane potential, was heat-stable, was inhibited by ruthenium red, lanthanides, monovalent cations, and certain anions. In vivo, when coupled to the protonmotive force, the systems would provide for the cell: (i) a mechanism to keep intracellular Ca2+ concentration low (Ca2+/2H+ antiporter); (ii) a mechanism for Ca2+ entry (electrogenic transporter).     

An electrogenic sodium pump in Limulus ventral photoreceptor cells

A hyperpolarization can be recorded intracellularly following either a single bright light stimulus or the intracellular injection of Na⁺. This after-hyperpolarization is abolished by bathing in 5 x 10^-6 M strophanthidin or removal of extracellular K⁺. Both treatments also lead to a small, rapid depolarization of the dark-adapted cell. When either treatment is prolonged, light responses can still be elicited, although with repetitive stimuli the responses are slowly and progressively diminished in size. The rate of diminution is greater for higher values of [Ca⁺⁺]_out; with [Ca⁺⁺]_out = 0.1 mM, there is almost no progressive diminution of repetitive responses produced by either K⁺-free seawater or strophanthidin. We propose that an electrogenic Na⁺ pump contributes directly to dark-adapted membrane voltage and also generates the after-hyperpolarizations, but does not directly generate the receptor potential. Inhibition of this pump leads to intracellular accumulation of sodium ions, which in turn leads to an increase in intracellular Ca⁺⁺ (provided there is sufficient extracellular Ca⁺⁺). This increase in intracellular calcium probably accounts for the progressive decrease in the size of the receptor potential seen when the pump is inhibited.