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PubMed2001

Glucose-induced oscillations in cytoplasmic free Ca2+ concentration precede oscillations in mitochondrial membrane potential in the pancreatic beta-cell.

Using dual excitation and fixed emission fluorescence microscopy, we were able to measure changes in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and mitochondrial membrane potential simultaneously in the pancreatic beta-cell. The beta-cells were exposed to a combination of the Ca(2+) indicator fura-2/AM and the indicator of mitochondrial membrane potential, rhodamine 123 (Rh123). Using simultaneous measurements of mitochondrial membrane potential and [Ca(2+)](i) during glucose stimulation, it was possible to measure the time lag between the onset of mitochondrial hyperpolarization and changes in [Ca(2+)](i). Glucose-induced oscillations in [Ca(2+)](i) were followed by transient depolarizations of mitochondrial membrane potential. These results are compatible with a model in which nadirs in [Ca(2+)](i) oscillations are generated by a transient, Ca(2+)-induced inhibition of mitochondrial metabolism resulting in a temporary fall in the cytoplasmic ATP/ADP ratio, opening of plasma membrane K(ATP) channels, repolarization of the plasma membrane, and thus transient closure of voltage-gated L-type Ca(2+) channels.

Adenosine Triphosphate↗
PubMed1987

Lymphocyte populations with different sensitivity to cyclosporin have different plasma membrane potentials.

Cyclosporin A (CsA), a clinically potent immunosuppressive agent, shows preferential biologic activity against certain lymphocyte subsets. To investigate this activity, we used flow cytometry to separate two distinct lymphocyte populations from unfractionated human peripheral blood lymphocytes, based on their different binding affinity for a biologically active fluorescent, dansylated cyclosporin (dans-CsA) derivative. The separate lymphocyte populations demonstrated different resting plasma membrane potentials. The two lymphocyte populations also had different levels of interleukin-2 (IL-2) receptors and shifted plasma membrane potential differently upon treatment with cyclosporin and lymphokine IL-2. The cell population that bound more dans-CsA contained the cells which possessed IL-2 receptors and responded to CsA and IL-2 with changes in membrane potential. The cell population that did not effectively bind dans-CsA lacked IL-2 receptors and did not respond to CsA or IL-2 with immediate membrane potential changes. Furthermore, stimulation studies of these separated two lymphocyte populations with phytohemagglutinin in the presence and absence of CsA revealed that only the population which binds a low level of dans-CsA shows a marked difference in membrane potential between the cells stimulated in the presence and absence of CsA. We concluded that ion flux changes caused by CsA affect the activation process of naive lymphocytes but not that of already stimulated ones. It can also be postulated that the ion flux changing property of CsA could constitute its primary or main mode of action.

Cell Membrane↗
PubMed2005

Simultaneous optical imaging of membrane potential and intracellular calcium.

Imaging of membrane potential (Vm) and intracellular calcium (Cai2+) is important for studying mechanisms of cardiac excitation, arrhythmias and defibrillation. We have developed an optical technique for simultaneous mapping of Vm and Cai2+ in cultured cell monolayers using fluorescent Vm- and Ca2+-sensitive dyes. Cultures of neonatal rat myocytes were double-stained with dyes RH-237 (Vm) and an analog of Fluo-3 or Rhod-2 dyes (Ca2+). These dyes have overlapping excitation spectra, allowing simultaneous excitation at the same wavelength range, and separate emission spectra allowing division of the fluorescent light into components sensitive to Vm and Cai2+, which were measured with two 16x16 photodiode arrays. It was found that Cai2+ measurements were strongly dependent on the properties of Ca2+-sensitive dyes. Thus, high-affinity Ca2+ dyes such as Fluo-4 and Rhod-2 reported Cai2+ transients approximately twice as long as those reported by low-affinity dyes Fluo-4FF and Rhod-FF. In addition, dyes with different affinities resulted in different measurements of Cai2+ responses to electrical shocks. When shocks were applied during the early plateau phase of the action potential, low-affinity dyes reported transient Cai2+ decreases at sites of both negative and positive Vm changes. In contrast, high-affinity Ca2+ dyes reported only a negligible change of plateau Cai2+ and a large elevation of diastolic Cai2+. These discrepancies between high- and low-affinity dyes were explained by a model of dye-ion interaction, which indicated that apparently long Cai2+ transients and Cai2+ responses to electrical shocks measured with high-affinity dyes were due to their non-linear response. These results indicate that optical measurements of Cai2+ transient duration and shock-induced Cai2+ changes require the use of low-affinity Cai2+ dyes.

Affinity Labels↗
PubMedDate not supplied

[Membrane potential levels in Acetabularia cells].

The membrane potential (MP) of A. mediterranea cell is shown to have two stationary levels RP==--170 mv and EP=--80 mV and an unstable one -- level of action potential peaks -- EP=--60 mV. The following transitions between the MP levels are possible: RP leads to EP' equilibrium EP leads to RP. The transition RP leads to EP' always has an exponential region. Values of the MP levels and the time constant of RP leads to EP' transition are equal in spontaneous AP, in AP induced by switching off the light or current pulse, when the cell is treated with 10(-3) M solution of DNP and when a part of cell is amputated. It is suggested that RP leads to EP' transition takes place when the electrogenic Cl-pump is switched off, while EP leads to RP when it is switched on.

Acetabularia↗
PubMed1999

Intrinsic theta-frequency membrane potential oscillations in hippocampal CA1 interneurons of stratum lacunosum-moleculare.

The ionic conductances underlying membrane potential oscillations of hippocampal CA1 interneurons located near the border between stratum lacunosum-moleculare and stratum radiatum (LM) were investigated using whole cell current-clamp recordings in rat hippocampal slices. At 22 degrees C, when LM cells were depolarized near spike threshold by current injection, 91% of cells displayed 2-5 Hz oscillations in membrane potential, which caused rhythmic firing. At 32 degrees C, mean oscillation frequency increased to 7.1 Hz. Oscillations were voltage dependent and were eliminated by hyperpolarizing cells 6-10 mV below spike threshold. Blockade of ionotropic glutamate and GABA synaptic transmission did not affect oscillations, indicating that they were not synaptically driven. Oscillations were eliminated by tetrodotoxin, suggesting that Na+ currents generate the depolarizing phase of oscillations. Oscillations were not affected by blocking Ca2+ currents with Cd2+ or Ca2+-free ACSF or by blocking the hyperpolarization-activated current (Ih) with Cs+. Both Ba2+ and a low concentration of 4-aminopyridine (4-AP) reduced oscillations but TEA did not. Theta-frequency oscillations were much less common in interneurons located in stratum oriens. Intrinsic membrane potential oscillations in LM cells of the CA1 region thus involve an interplay between inward Na+ currents and outward K+ currents sensitive to Ba2+ and 4-AP. These oscillations may participate in rhythmic inhibition and synchronization of pyramidal neurons during theta activity in vivo.

Animals↗
PubMed1987

Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes.

A number of cellular functions have recently been associated with alterations of the membrane potential in non-excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage-sensitive oxonol dye di-BA-C4(3) was developed. The fluorescent signal of di-BA-C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH-sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the "putative" rat osteoblasts (UMR 106) as -28.3 +/- 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 +/- 0.7 mM (n = 3) and 24 +/- 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.

Animals↗
PubMed2006

[Optical mapping of the membrane potential with voltage-sensitive dyes].

Optical mapping of the membrane potential with voltage-sensitive dyes is an advanced approach that involves in many theories such as molecular photonica, physiology and computer science. Compared with the conventional techniques of membrane patch-clamp and microelectrode, the optical mapping system can measure not only the changes of a single membrane potential from multiple sites simultaneously but also the conduction properties of the cell populations. So this technique provides an important method for studying the electrophysiology of the small cell, the tiny neurite and the cardiac arrhythmia, etc. Because this technique can also avoid the electromagnetic interferences, it uniquely provides an ideal means for studying the mechanisms such as cardiac defibrillation. In this paper the principles and the system structure of the optical mapping are introduced, its applications and future developments are also presented at the same time.

Animals↗
PubMed2006

Mitochondrial metabolic states and membrane potential modulate mtNOS activity.

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.

Animals↗
PubMed1985

Changes in membrane potential and potassium and sodium activities during postnatal development of mouse skeletal muscle.

Dual-channel potassium-selective and single-channel sodium-selective microelectrodes were used to investigate the cause of changes in resting membrane potential of muscle fibers of the mouse during early development. The resting membrane of extensor digitorum longus fibers hyperpolarized during the postnatal period from -41.8 mV at 4 days of age to -76.4 mV at 27 days. During this period intracellular potassium activity increased by 42.1% from 82.5 mM at 8 days to 117.2 mM at 29 days. Intracellular sodium activity was high at 8 days, 23.7 mM, but decreased rapidly to adult values by 27 days when it was 9.98 mM, a 57.9% reduction in sodium activity. The time course of the change in resting membrane potential was different from that of the potassium equilibrium potential calculated from the data. If only potassium and sodium ions were to make significant contributions to the potential, then it was calculated that the permeability ratio PNa:PK would have to change from a value of 0.0659 at 8 days to 0.0227 at 27 days. The results indicated that other factors might be involved in generating the membrane potential inasmuch as, although both intracellular potassium and sodium activities did not change significantly after 27 to 30 days, the membrane potential had not attained adult values at that time. The possibility that increases in muscle activity during the postnatal period might initiate the changes in membrane polarization and intracellular ion activities is discussed together with possible complications in interpretation due to great variations in fiber diameters.

Animals↗
PubMed1992

Membrane potential modulates divalent cation entry in rat parotid acini.

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.

Animals↗
PubMed1991

Contributions of secondary active transport processes to membrane potentials.

Equations are developed to examine the effects of secondary active transport processes on the steady-state membrane potential of symmetrical cells. It is shown that, with suitable modifications, equations of the type developed by Goldman, Hodgkin and Katz may be derived to accommodate the contributions to the membrane potential of both electroneutral and electrogenic transporters. Where the membrane potential is a function of the dominant medium ions (Na, K, and Cl), other contributions can come only from an electrogenic Na pump and from neutral co- and counter-transporters if, and only if, these involve the dominant ions. Experimental approaches to measure the parameters necessary to solve the equations developed here are discussed.

Animals↗
PubMed1990

Energetics of Ehrlich ascites mitochondria: membrane potential of isolated mitochondria and mitochondria within digitonin-permeabilized cells.

Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.

Animals↗
PubMed2006

Quantitative techniques for steady-state calculation and dynamic integrated modelling of membrane potential and intracellular ion concentrations.

The membrane potential (E(m)) is a fundamental cellular parameter that is primarily determined by the transmembrane permeabilities and concentration gradients of various ions. However, ion gradients are themselves profoundly influenced by E(m) due to its influence upon transmembrane ion fluxes and cell volume (V(c)). These interrelationships between E(m), V(c) and intracellular ion concentrations make computational modelling useful or necessary in order to guide experimentation and to achieve an integrated understanding of experimental data, particularly in complex, dynamic, multi-compartment systems such as skeletal and cardiac myocytes. A variety of quantitative techniques exist that may assist such understanding, from classical approaches such as the Goldman-Hodgkin-Katz equation and the Gibbs-Donnan equilibrium, to more recent "current-summing" models as exemplified by cardiac myocyte models including those of DiFrancesco & Noble, Luo & Rudy and Puglisi & Bers, or the "charge-difference" modelling technique of Fraser & Huang so far applied to skeletal muscle. In general, the classical approaches provide useful and important insights into the relationships between E(m), V(c) and intracellular ion concentrations at steady state, providing their core assumptions are fully understood, while the more recent techniques permit the modelling of changing values of E(m), V(c) and intracellular ion concentrations. The present work therefore reviews the various approaches that may be used to calculate E(m), V(c) and intracellular ion concentrations with the aim of establishing the requirements for an integrated model that can both simulate dynamic systems and recapitulate the key findings of classical techniques regarding the cellular steady state. At a time when the number of cellular models is increasing at an unprecedented rate, it is hoped that this article will provide a useful and critical analysis of the mathematical techniques fundamental to each of them.

Animals↗
PubMed1990

Correlation between plasma membrane potential and second messenger generation in the promyelocytic cell line HL-60.

The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.

Calcium↗
PubMed1978

Reduction of membrane potential, an immediate effect of colicin K.

Colicin K causes a rapid and drastic reduction of the membrane potential of Escherichia coli cells, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium. The colicin causes no major changes in the pH gradient, as measured by the uptake of butyric acid. The decrease in membrane potential following addition of colicin K to the cells is a prompt response that parallels or precedes known physiological effects such as efflux of accumulated substrates. Hence the loss of membrane potential qualifies as the primary action by which the colicin uncouples membrane-associated function from respiration. Certain peculiarities of bacterial cells pretreated with EDTA in their response to uncoupling agents and lipophilic ions are described.

Biological Transport↗
PubMed1989

Lymphocyte subpopulation with low membrane potential in the blood of cyclosporin- and prednisone-treated patients: in vivo selectivity for T4 subset.

Previous work on the mode of action of CsA revealed that this drug shifts the membrane potential of human periferal blood lymphocytes in vitro. Recently we have analyzed lymphocytes of cyclosporin-treated transplant patients. Membrane potential analysis with the DIOC6(3) fluorescence dye indicates that all the studied patients have a subpopulation of lymphocytes with low membrane potential and that this population is made up predominently of OKT4+ cells. However there is no correlation between the clinical events and the percentage distribution of lymphocyte subpopulations as defined by the membrane potential and the T4/T8 ratio of the total lymphocyte population.

CD4-Positive T-Lymphocytes↗
PubMed1990

Membrane potential of Plasmodium falciparum gametocytes monitored with rhodamine 123.

The membrane potential of Plasmodium falciparum gametocytes was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123) as a probe. Epifluorescence microscopy revealed that R123 at 1 microgram/ml rather selectively partitioned into structure resembling large mitochondria. Treatment of R123-loaded gametocytes with various inhibitors including those of respiration resulted in disappearance of fluorescence from what appeared to be the mitochondria, but not from the cytosol. These results indicate that P. falciparum gametocytes have the mitochondrion maintaining an inside negative membrane potential.

Animals↗
PubMed1998

[Effects of hypertonic solution on the membrane potential of isolated outer hair cell in the guinea pig cochlear].

To learn effects of hypertonic solution on the membrane potential and cell length of isolated outer hair cell in the guinea pig cochlear, we determined the membrane potential and cell length during hypertonic activation with ACAS 570 and the membrane potential sensitive probe DiBAC4(3). The results showed that the hypertonic activation depolarized the outer hair cell and made it elongate. We think that the outer hair cell has activity, the depolarization and elongation of the outer hair cell caused by hypertonic solution are cyto-electrophysiological basis of hearing loss and recruitment in Meniere's disease.

Animals↗