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PubMed1999

Determination of the individual electrical and transport properties of the plasmalemma and the tonoplast of the giant marine alga Ventricaria ventricosa by means of the integrated perfusion/charge-pulse technique: evidence for a multifolded tonoplast.

The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane resistance of the tonoplast of these marine giant algal cells is apparently very high as reported already for V. utricularis. The exceptionally high value of the area-specific capacitance could be explained - among other interpretations - by assuming a 9-fold enlargement of the tonoplast surface. The hypothesis of a multifolded tonoplast was supported by transmission electronmicroscopy of cells fixed under maintenance of turgor pressure and of the electrical parameters of the membranes. This finding indicates that the tonoplast of this species exhibited a sponge-like appearance. Taking this result into account, it can be easily shown that the tonoplast exhibits a high-resistance (1.1 Omega m2). Vacuolar membrane potential measurements (performed in parallel with charge-pulse relaxation studies) showed that the potential difference across the plasmalemma was mainly controlled by the external K+-concentration which suggested that the resting membrane potential of the plasmalemma is largely a K+-diffusion potential. After permeabilization of the tonoplast with nystatin the potential of the intact membrane barrier dropped from about slightly negative or positive (-5.1 to +18 mV, n = 13) to negative values (-15 up to -68 mV; n = 8). This indicated that the cytoplasm of V. ventricosa was apparently negatively charged relative to the external medium. Permeabilization of the plasmalemma by addition of external nystatin resulted generally in an increase in the potential to slightly more positive values (-0.8 to +4.3 mV; n = 5), indicating that the vacuole is positively charged relative to the cytoplasm. These findings apparently end the long-term debate about the electrical properties of V. ventricosa. The results presented here support the findings of Davis (Plant Physiol. 67: 825-831, 1981), but are contrary to the results of Lainson and Field (J. Membrane Biol. 29: 81-94, 1976).

Action Potentials↗
PubMed1992

Modeling the circulation with three-terminal electrical networks containing special nonlinear capacitors.

Development, first of analog and later of digital computers, as well as algorithms for analysis of electrical circuits, stimulated the use of electrical circuits for modeling the circulation. The networks used as building blocks for electrical models can provide accurate representation of the hydrodynamic equations relating the inflow and outflow of individual segments of the circulation. These networks, however, can contain connections in which voltages and currents have no analogues in the circulation. Problems arise because (a) electrical current must flow in closed loops, whereas no such constraints exist for hydraulic models; and (b) electrical capacitors have a number of characteristics that are not analogous to those of hydraulic compliant chambers. Disregarding these differences can lead to erroneous results and misinterpretation of phenomena. To ensure against these errors, we introduce an imaginary electrical element, the nonlinear residual-charge capacitor (NRCC), with characteristics equivalent to those of a compliant chamber. If one uses appropriate circuit connections and incorporates the residual-charge capacitor, then all voltages and currents in the model are proper analogues of pressures and flows in the circulation. It is shown that the capacitive current represents the rate of change of volume of blood inside the vessel, as well as the rate of the corresponding displacement of volume of the surrounding tissue.

Blood Circulation↗
PubMed1978

Effect of polymyxin B on the structure and the stability of lipid layers.

Polymyxin B (PX) does not penetrate phospholipid monolayers and bilayers at low field strength across the lipid layers. The degree of penetration of PX is evaluated from its effect on the capacitance of the monolayers and on the conductance of the bilayers. PX added to one side of a bilayer causes its destabilization, it also enhances destabilization of lipid monolayers at positive electric fields across the surface layer in the direction of the adsorbed PX. PX lowers very little the fluorescence polarization of 1,6-diphenyl 1,3,5 hexatriene embedded in phospholipid vesicles. It is suggested that the penetration mechanism of PX into gram-negative bacteria is based on transient local breakdown of the plasma membrane.

Adsorption↗
PubMed2001

Electrostatic surface plasmon resonance: direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches.

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

Base Pair Mismatch↗
PubMed1986

Cardiopulmonary resuscitation with interposed abdominal compression.

The addition of interposed abdominal compressions (IACs) to otherwise standard CPR enhances artificial circulation both in anesthetized dogs with ventricular fibrillation and in electrical models of the circulation that demonstrate fundamental mechanisms generating flow. Manual abdominal compressions cause both central aortic and central venous pressure pulses but, because of differences in venous and arterial capacitance, the former are usually greater than the latter. Thus mean perfusion pressure is enhanced. Limited clinical studies confirm that IAC-CPR can improve perfusion pressures in humans, and reported complications of the technique are rare in animals and man. However, no study has demonstrated that IAC-CPR improves either short- or long-term survival after cardiac arrest in man. Accordingly, the method remains experimental and cannot be recommended for basic life support at the present time.

Abdomen↗
PubMed2001

The effects of interpulse interval on stochastic properties of electrical stimulation: models and measurements.

It is known that some cochlear implant users have improved speech perception using higher rates of interleaved pulsatile stimulation. There are, however, significant limitations on their performance presumably due in part to temporal and spatial interactions. To address these limitations, we have examined refractory characteristics of the auditory nerve using experimental animal models and computational simulations. A stochastic model of the node of Ranvier modified for mammalian sodium channel kinetics has been developed to calculate the masked input-output (I/O) functions for different interpulse intervals (IPI) [26]. The model is based upon 1000 voltage-gated sodium channels and incorporates parameters such as nodal resistance and capacitance. The relative spread (RS) [35] calculated from the I/O functions was typically 0.03 for 17 different IPIs between 450 micros and 6 ms for cathodal stimuli. For IPI = 830 and 870 micros, the RS was ten times greater than those for other IPIs. Although it is not fully understood how the electrically evoked compound action potential (EAP) data are related to single fiber data, the RS of single fibers is a partial contributor [19]. We have measured the EAP using a monopolar intracochlear stimulating electrode and a recording electrode placed directly on the nerve and have observed changes in slope of EAP growth functions consistent with the theoretical RS values. These results have significant implications for speech coding in a cochlear implant since they suggest an increased membrane noise for pulse trains of specific rates.

Action Potentials↗
PubMed1974

Linear analysis of membrane conductance and capacitance in cardiac Purkinje fibres.

1. The membrane capacitance and slope conductance of sheep cardiac Purkinje tissue were examined under conditions of constant and slowly changing voltage within the potential range: -70 to -100 mV. Data were secured by applying constant current pulses, or by imposing ramp commands or small changes in holding potential under voltage clamp control.2. The electrical measurements, combined with light and electron microscopic data on preparation structure, were interpreted with the aid of a core-conductor model that permits quantitative representation of cleft- and lateral surface membranes for individual preparations (Hellam & Studt, 1974).3. The analysis led to the following conclusions. The slope conductance of unit membrane area (G(m)) is quite low in the normal resting potential range; at -80 mV the value is 0.09 mmho/cm(2). G(m) exhibits a marked decline with depolarization in the region positive to -88 mV, but only slight voltage-dependence at more negative potentials within the range of present observations. The capacitance of unit membrane area (C(m)), from voltage ramp data collected near -90 mV, is 0.9 muF/cm(2).4. Comparison with results of earlier studies indicates that the differences between present and previous experimental estimates of these membrane characteristics result from differences in modelling and structural analysis of tissue preparations.

Animals↗
PubMed2005

Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation.

Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.

Cell Membrane↗
PubMed2007

Synthesis and properties of trigeminal tricationic ionic liquids.

Novel trigeminal tricationic ionic liquids (TTILs) have been successfully synthesized in high yields by means of Menschutkin quaternization via an S(N)1 mechanism. This reaction presents a new convenient method for transforming glycerol into multifunctional compounds. The physical properties of a series of TTILs were characterized by using a variety of techniques. The prepared salts were tested for antimicrobial activity. Electrochemical characterization of TTILs was also performed, which allowed the estimation of the conductivity of these new compounds, to establish their electrochemical stability window and capacitance properties over a wide range of temperatures. A good correlation of the physical properties of TTILs with capacitance values was observed.

Anti-Bacterial Agents↗
PubMed1997

The outer hair cell motor in membrane patches.

We have used patch-clamp techniques to record the charge movement associated with motility in patches of basolateral membrane from isolated outer hair cells. Charge movement has been measured from the voltage-dependent capacitance. Using 3 to 4 Momega pipettes with tip diameters of 3 micron the measured maximum voltage-dependent capacitance was 56 +/- 6 fF at -36 mV when the resting membrane potential was -20 mV. The calculated total charge movement was 5.6 +/- 0.6 fC (n = 13) and the inferred density of univalent motor elements was 8400/micron2. Negative pressure (applied via the pipette) increased membrane tension and shifted the capacitance peak to depolarised potentials. Under conditions of isotropic membrane stress there was no change in the peak measured capacitance in contrast to that measured in previous whole-cell recordings.

Animals↗
PubMed1999

Frequency-dependent capacitance of the apical membrane of frog skin: dielectric relaxation processes.

Impedance analysis of the isolated epithelium of frog skin (northern Rana pipiens) was carried out in the frequency range between 0.1 Hz and 5.5 kHz while Na+ transport was abolished. Under these conditions, the impedance is determined almost completely by the dielectric properties of the apical membranes of the cells and the parallel shunt resistance. The modeling of the apical membrane impedance function required the inclusion of dielectric relaxation processes as originally described by. J. Chem. Phys. 9:341-351), where each process is characterized by a dielectric increment, relaxation frequency, and power law dependence. We found that the apical plasma membrane exhibited several populations of audio frequency dielectric relaxation processes centered at 30, 103, 2364, and 6604 Hz, with mean capacitive increments of 0.72, 1.00, 0.88, and 0.29 microF/cm2, respectively, that gave rise to dc capacitances of 1.95 +/- 0.06 microF/cm2 in 49 tissues. Capacitance was uncorrelated with large ranges of parallel shunt resistance and was not changed appreciably within minutes by K+ depolarization and hence a decrease in basolateral membrane resistance. A significant linear correlation existed between the dc capacitance and Na+ transport rates measured as short-circuit currents (Cadc = 0.028 Isc + 1.48; Isc between 4 and 35 microA/cm2) before inhibition of transport by amiloride and substitution of all Na+ with NMDG (N-methyl-D-glucamine) in the apical solution. The existence of dominant audio frequency capacitive relaxation processes complicates and precludes unequivocal interpretation of changes of capacitance in terms of membrane area alone when capacitance is measured at audio frequencies.

Animals↗
PubMed2001

Linoleamide, a brain lipid that induces sleep, increases cytosolic Ca2+ levels in MDCK renal tubular cells.

Linoleamide is an endogenous lipid that has been shown to induce sleep in cats, rats and humans. However, its physiological function remains unclear. In this study the effect of linoleamide on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) tubular cells was examined, by using fura-2 as a Ca2+ probe. In a concentration-dependent manner, linoleamide induced increases in [Ca2+]i between 10-500 microM with an EC50 of 20 microM. The signal comprised a slow rise and a persistent phase, and was a result of internal Ca2+ release and external Ca2+ influx because it was partly inhibited by external Ca2+ removal. In Ca2+-free medium, depletion of the endoplasmic reticulum Ca2+ store with 1 microM thapsigargin abolished 100 microM linoleamide-induced internal Ca2+ release, and conversely, pretreatment with linoleamide prevented thapsigargin from releasing internal Ca2+. This demonstrates that the internal source of linoleamide-induced [Ca2+]i increase is located in the endoplasmic reticulum. This discharge of internal Ca2+ caused capacitative Ca2+ entry because after incubation with 100 microM linoleamide in Ca2+-free medium for 8 min readmission of 3 mM CaCl2 induced increases in [Ca2+]i. After the formation of inositol-1,4,5-trisphosphate (IP3) was blocked by the phospholipase C inhibitor U73122 (1 microM), linoleamide still induced an increase in [Ca2+]i but the shape of the increase was altered. Similar results were found for another sleep-inducing lipid 9,10-octadecenoamide. Together, the present study shows that the endogenous sleep-inducing lipid linoleamide was able to cause significant increases in [Ca2+]i in renal tubular cells, by releasing the endoplasmic reticulum Ca2+ store and triggering capacitative Ca2+ entry in a manner independent of IP3.

Animals↗
PubMed1995

Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels.

Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.

Calcium↗
PubMed1992

Comparison of impedance at the microelectrode-saline and microelectrode-culture medium interface.

The impedance characteristics of gold-plated indium-tin-oxide microelectrodes immersed in culture medium (MEM) are described and compared with the impedance characteristics observed when those microelectrodes are immersed in isotonic saline. For microelectrode areas of approximately 100 microns2, applied voltage levels of 5, 50, and 100 mV, and for frequencies of from 100 Hz to 10 kHz the resistance, capacitance, capacitive reactance, and total impedance are given as a function of frequency both in culture medium and in saline. The results, which hold for current densities ranging from 0.45 to 700 pA/microns2, are compared. Also given are the alpha and K values determining the frequency characteristics of the interface resistance and capacitance in medium and in saline.

Culture Media↗
PubMed1976

A non-linear voltage dependent charge movement in frog skeletal muscle.

1. Voltage-clamp experiments were carried out using the three microelectrode technique. Using this method membrane current density at V1 is proportional to deltaV( = V2 - V1) where V1 and V2 are voltages at distances 1 and 21 from the end of a fibre. Voltage dependent sodium currents were blocked by tetrodotoxin, potassium by tetraethylammonium ions and rubidium. Contraction was blocked by adding sucrose, 467 mM. 2. The current deltaV (control) associated with a positive voltage step from a hyperpolarized conditioning voltage to the holding potential, -80 mV, showed two components, a capacitative transient which decayed rapidly and a maintained steady level...

Animals↗
PubMed1981

Membrane capacitance in hyperpolarized muscle fibres.

1. Voltage clamp experiments on muscle fibres compared transients to 10 mV steps at hyperpolarized voltages between VT = -100 to -185 mV with controls obtained at VC = -85 mV. 2. Membrane capacitance fell by 15%, 13% and 4.5% per 100 mV hyperpolarization fro -85 mV in hypertonic low chloride, hypertonic high chloride and isotonic high chloride-containing solutions respectively. 3. The charge moved by the 'on' and 'off' parts of the applied step was equal over the voltages studied. This suggests that the changes were capacitative rather than ionic in origin. The changes could not be explained in terms of the cable properties of the transverse tubular system. They may therefore reflect non-linear capacitance in the muscle membrane itself. 4. Subtracting control transients at -85 mV from transients obtained at different voltages gave monotonically decaying charge movements. In the frequency domain, these charge movements possessed real and imaginary permittivities resembling those of a 'Debye' particle. 5. It is concluded that muscle membranes have non-linear capacitances even at voltages far hyperpolarized to those in which familiar voltage-dependent processes occur.

Animals↗
PubMed1967

Frog skeletal muscle fibers: changes in electrical properties after disruption of transverse tubular system.

In muscle fibers which have been exposed for 1 hour to a Ringer solution containing 400 millimolar glycerol and then returned to plain Ringer solution, the transverse tubular system is disrupted. At the same time the membrane capacitance is markedly reduced and hyperpolarizing current pulses no longer produce a slow, progressive increase in potential (creep). The large capacitance of muscle and the phenomenon of "creep" must both depend on an intact transverse tubular system.

Animals↗