Calcium bursts induced by nanosecond electric pulses

We report here real-time imaging of calcium bursts in human lymphocytes exposed to nanosecond, megavolt-per-meter pulsed electric fields. Ultra-short (less than 30 ns), high-field (greater than 1 MV/m), electric pulses induce increases in cytosolic calcium concentration and translocation of phosphatidylserine (PS) to the outer layer of the plasma membrane in Jurkat T lymphoblasts. Pulse-induced calcium bursts occur within milliseconds and PS externalization within minutes. Caspase activation and other indicators of apoptosis follow these initial symptoms of nanosecond pulse exposure. Pulse-induced PS translocation is observed even in the presence of caspase inhibitors. Ultra-short, high-field, electroperturbative pulse effects differ substantially from those associated with electroporation, where pulses of a few tens of kilovolts-per-meter lasting a few tens of microseconds open pores in the cytoplasmic membrane. Nanosecond pulsed electric fields, because their duration is less than the plasma membrane charging time, develop voltages across intracellular structures without porating the cell.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Single base mismatch detection by microsecond voltage pulses

A single square voltage pulse applied to metal electrodes underneath a silicon dioxide film upon which DNA probes are immobilized allows the discrimination of DNA targets with a single base mismatch during hybridization. Pulse duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of electric field assisted hybridization. Although pulses with 1 V, lasting less than 1 ms and with a rise/fall times of 4.5 ns led to maximum hybridization of fully complementary strands, lack of stringency did not allow the discrimination of single base mismatches. However, by choosing pulse conditions that are slightly off the optimum, the selectivity for discriminating single base mismatches could be improved up to a factor approximately 5 when the mismatch was in the middle of the strand and up to approximately 1.5 when the mismatch was on the 5'-end and. These results demonstrate that hybridization with the appropriate electric field pulse provides a new, site-specific, approach to the discrimination of single nucleotide polymorphisms in the sub-millisecond time scale, for addressable DNA microarrays.     

Calcium waves in astrocytes-filling in the gaps.

Stimulus-evoked cellular responses are sometimes organized in the form of propagating waves of cytoplasmic Ca2+ increase. Ca2+ waves can be elicited in cultured astrocytes by the neurotransmitter glutamate; however, the propagation mechanism is unknown. Here, qualitative and quantitative features of propagation suggest that astrocytic Ca2+ waves are mediated by an intracellular signal that crosses intercellular junctions. The role of gap junctions in cell-cell Ca2+ wave propagation was specifically tested. Functional gap junctions were demonstrated using a noninvasive fluorescence recovery method and the gap junction blockers halothane and octanol. Gap junction closure prevented intracellular waves from propagating between cells without affecting the velocity of the intracellular wave itself. The pivotal role played by the gap junction creates the potential for dynamic changes in glial connectivity and long-range glial signaling.     

Rapid changes in flagellar rotation induced by external electric pulses.

The bacterial flagellar motor is the only molecular rotary machine found in living organisms, converting the protonmotive force, i.e., the membrane voltage and proton gradients across the cell membrane, into the mechanical force of rotation (torque). We have developed a method for holding a bacterial cell at the tip of a glass micropipette and applying electric pulses through the micropipette. This method has enabled us to observe the dynamical responses of flagellar rotation to electric pulses that change the membrane voltage transiently and repeatedly. We have observed that acceleration and deceleration of motor rotation are induced by application of these electric pulses. The change in the rotation rate occurred within 5 ms after pulse application.     

Chloroplast translocations induced by light pulses

The effect of single blue-light pulses on chloroplast rearrangement was studied in the leaves of Tradescantia albiflora, Chlorophytum elatum, and Lemna trisulca. For measuring translocations in terrestrial plants the method of transmission changes was used; translocations in the water plant Lemna were studied by direct microscopic observation and counting. Strong light (30 W m^-2) applied in the form of short pulses, shorter than a lag period of translocations, induces some transient effects in the following dark period. With short pulses, transient rearrangements of chloroplasts to a weak-light position were found. With longer pulse duration, biphasic responses took place in Tradescantia and Lemna: The initial movement to a partial strong-light position was followed by a wave of translocation to a weak-light arrangement. In Chlorophytum this type of response appeared only in a narrow fluence range. The validity of the reciprocity law in relation to fluence rate and time of irradiation was confirmed for Tradescantia. The results may give us an insight into the kinetics of the primary effects of light in the translocation process.     

Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

Calcium ion uptake in isolated pancreas cells induced by secretagogues.

1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein and the Ca2+ ionophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3',5'-monophosphate and dibutyrylic cyclic guanosine 3',5'-monophosphate have no effect on 45Ca uptake. 2. A graphical analysis of the Ca2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca2+ to the membrane and a slow phase representing Ca2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. The 45Ca-exchangeable pool size is increased by both carbamylcholine and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca2+ transfer into the cell most probably through an increase of the cell membrane permeability for Ca2+.     

Chloroplast translocations in Lemna trisulca L. induced by continuous irradiation and by light pulses

The analytical model describing the steady state position of chloroplasts in dependence of fluence rate as well as the chloroplast response to single strong light pulses has been proposed. The model is based on the following assumptions: 1. Irradiation of the cell generates the state X in the cell membrane region, proportional to the local fluence rate. After switching on the light, the value of X increases exponentially with the time constant of about 3 min. The dark decay of X is also exponential with the same time constant. The level of X controls all kinds of chloroplast arrangements. 2. The state X generates two further states: Y ₁ and Y ₂, the first of them representing attraction forces for chloroplasts and the second representing repulsion forces. Empirical equations have been found for both Y states. The fluence rate response curve can be described with the use of functions Y ₁ and Y ₂. 3. The kinetic analysis requires the introduction of two additional functions Z in order to account for delays and time dispersion of the chloroplast movement in response to driving and resistance factors. The computer program for the proposed model was developed and the results of calculations were compared with experimental data (fluence rate response curve and pulse effects) with satisfactory agreement. Initially no attempt was made to ascribe any physical meaning to the postulated states. Some suggestions in this respect are mentioned in the discussion.     

Calcium ion independent membrane leakage induced by phospholipase-like myotoxins.

The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.     

Calcium waves and oscillations in eggs

Eggs from several protostomes (molluscs, annelids, nemerteans, etc.) and two deuterostomes (mammals and ascidians) display repetitive calcium signals. Oscillations in the level of intracellular calcium concentration are occasionally triggered by maturing hormones (as in some molluscs) and mostly observed after fertilization which occurs at different stages of the meiotic cell cycle (oocytes are arrested in prophase, metaphase I or metaphase II). In most eggs examined so far, calcium oscillations last until the end of meiosis just before male and female pronuclei form. This ability depends on the sensitivity of InsP3 channels and on the permeability of the plasma membrane to extracellular calcium. In eggs that undergo cytoplasmic reorganization at fertilization (annelids, nemerteans, ascidians, etc.) the repetitive calcium signals are waves that originate from localized cortical sites that become calcium waves pacemakers. In ascidians we have identified the site of initiation of repetitive calcium waves as an accumulation of endoplasmic reticulum sandwiched between the plasma membrane and an accumulation of mitochondria. We compare and discuss the generation of calcium signals in the different eggs, their relationship with the cell cycle and the possible roles they play during development.     

Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

Calcium oscillations induced by gambierol in cerebellar granule cells

Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage‐dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 µM, gambierol‐evoked [Ca²⁺]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol‐evoked [Ca²⁺]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K⁺ channel blocker 4‐aminopyridine (4‐AP)‐evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4‐AP and indicate that gambierol‐induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage‐dependent potassium channels and hyperpolarization of sodium current activation. J. Cell. Biochem. 110: 497–508, 2010. © 2010 Wiley‐Liss, Inc.     

Solitary waves in soybean induced by localized thermal stress

Action potentials in higher plants are believed to be the information carriers in intercellular and intracellular communication in the presence of an environmental stressor. Plant electrophysiologists have recorded long distance electrical signaling in higher plants during the last two hundred years. Reproducing the duration, speed of propagation, and the shape of the action potential is challenging. Early measurements revealed that the speed of action potential propagation in plants is extremely slow - from 0.1 mm/s to 20 cm/s, although many faster plant responses to stress have been recorded as well. We hypothesized that this discrepancy is most likely due to the artifacts of aliasing from slow registration systems. In this study, we employ real time measurements using modern data acquisition techniques to detect ultra fast action potentials in green plants induced by localized heat stress. Thermal shock or heat stress is the most common environmental stress. Based on more sophisticated measuring techniques, we show that plants transmit solitary waves and that the speed of action potential propagation in green plants is similar to the speed of action potentials in mammalians, varying from a few meters per second up to 105 m/s. Possible pathways for electrical signal propagation in vascular plants are discussed.Key words: Action potential, plant electrophysiology, electrical signaling, localized heat stress, excitability     

Alterations induced by large doses of furosemide in chronic renal insufficiency.

Eleven patients with different degrees of renal failure with creatinine clearances between 7 and 32 ml/min have been studied. After a standard water overload and control periods of clearances, furosemide 1 g was given/i.v. There followed significant increase of renal plasma flow and glomerular filtration rate. In one case the increase was maintained during a follow up period of 3 hours. A significant increase was evident in phosphate, uric acid, sodium, potassium, and calcium clearances, as well as an increase in the sodium delivery to the distal nephron and a decrease in tubular reabsorption of phosphate. All this may be interpreted as the result of renal vasodilation induced by furosemide and its effect upon the proximal tubule and on Henle's loop.