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At least 811 records · Page 45Linked to original sources
PubMed1997

Measurement of membrane potential in Saccharomyces cerevisiae by the electrochromic probe di-4-ANEPPS: effect of intracellular probe distribution.

Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane. The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time. The presence and properties of the cell wall affected the extent of cell staining. The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy. The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min. Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

Cell Membrane↗
PubMed1983

The basis for the membrane potential of quiescent cells of the canine coronary sinus.

During prolonged periods of quiescence, the membrane potential of cells in the isolated canine coronary sinus, exposed to normal Tyrode solution containing 4 mM-K, declines to about -60 mV. The nature of the resting potential was investigated, in small strips of coronary sinus tissue mounted in a fast-flow system, by recording the membrane potential responses to sudden changes in the extracellular ionic environment. At extracellular K concentrations ([K]o) from 0 to 64 mM the resting potential was little affected by replacing all but 1 mM of external Cl ions with isethionate and methylsulphate ions. At [K]o levels from 4 to 150 mM the resting potential was reasonably well described by the Goldman-Hodgkin-Katz equation on the assumption that the intracellular K concentration ([K]i) was 155 mM and that the ratio of membrane permeability coefficients for Na and K, PNa/PK, was 0.07. In the presence of a high concentration of acetylcholine or carbachol (greater than or equal to 1 microM), the resting potentials at [K]o levels from 1 to 150 mM approximated K equilibrium potentials (EK) calculated on the assumption that [K]i was 155 mM. At [K]o levels less than or equal to 8 mM replacing most of the external Na with sucrose or Tris caused a substantial hyperpolarization, whereas application of 1-2 microM-tetrodotoxin caused only slight hyperpolarization. A transient hyperpolarization, due to enhanced electrogenic Na extrusion, was recorded on switching back to 4 mM-K following brief exposures to K-free solution; no transient hyperpolarization was recorded in the presence of 5 microM-acetylstrophanthidin. The acetylstrophanthidin itself caused a rapid depolarization of several millivolts. Preliminary conductance measurements made with two micro-electrodes in some smaller preparations indicate that the steady-state current-voltage relationship is N-shaped. We conclude that the low membrane potential of quiescent coronary sinus cells reflects not a low [K]i but rather a relatively high ratio PNa/PK, of about 0.07: the Na ions flow into the cells via predominantly TTX-insensitive pathways and are extruded by the electrogenic Na/K exchange pump, which thereby makes a substantial contribution to the resting potential.

Acetylcholine↗
PubMed1985

Effect of membrane potential on the kinetic parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles.

Comparison of the transport properties of the melibiose permease of E. coli acting as a H+-symport or a Na+-symport has been performed by measuring initial rates of [3H]-melibiose transport or its accumulation in isolated membrane vesicles. The results show strikingly that although the membrane potential primarily drives melibiose accumulation by both types of symport, it selectively affects the apparent affinity constant Kt of the H+-melibiose symport while it specifically changes the maximal rate of transport (Vmax) of the Na+-melibiose symport. It is suggested that modification(s) of some partial reaction constants of a given transport cycle might lead to important changes in the kinetic properties of this transport system.

Biological Transport, Active↗
PubMed1982

Cellular membrane potentials and contractile threshold in mammalian skeletal muscle susceptible to malignant hyperthermia.

An increase in external K+ can generate contractile force in muscle, and K+ contractures are an established means to determine the effect of an agent on the relation between membrane potential and mechanical activity [8]. We have used K+-contractures to further test our hypothesis [5] that abnormal cell membrane potential responses are intrinsic to skeletal muscle of Poland China pigs susceptible to malignant hyperthermia (MHS), and a nervous system is not required to initiate malignant hyperthermia (MH). Recently it has been shown that K+-induced contractures in certain skeletal and cardiac muscles might not be determined only by cellular membrane potential changes predicted by the tendency for K+ to move in accord with its electrical and concentration gradients [3, 12]. We report here that normal and diseased skeletal muscles respond differently to raised external K+, which supports the idea that the onset of the MH syndrome is determined by a defect associated with a site in muscle cells superficial to the cytoplasm rather than, as has been suggested, within the cell or in the nervous system [7]. In addition, we find that porcine skeletal muscles, like certain other muscles, produce much less force in K+ than expected from our measurements of the relationship between membrane potential and external K+.

Animals↗
PubMed2001

A microfluidic device for measuring cellular membrane potential.

Recent developments in microfluidics have enabled the design of a lab-on-a-chip system capable of measuring cellular membrane potential. The chip accesses liquid samples sequentially by sipping from a microplate through a capillary, mixes the samples with cells flowing through a microchannel, contacts the cells with potential-sensitive dyes, and reads out cellular responses using fluorescence detection. The rate of cellular uptake of membrane-permeable, ionic fluorophores by THP-1 cells was found to depend strongly on membrane potential. The ratio of the fluorescence of the anionic dye DiBAC(4)(3) and the cationic dye Syto 62 taken up by cells was found to double for every 33 mV change in membrane potential. The utility of this approach was demonstrated by assaying ion channel activity in human T lymphocytes. Because of the high sensitivity, low cellular and reagent consumption, and high data quality obtained with the microfluidic device, the lab-on-a-chip system should be widely applicable in high-throughput screening and functional genomics studies.

Barbiturates↗
PubMed1998

Optical recording of membrane potential on isolated spiral ganglion cells of newborn mice using a voltage-sensitive dye.

Optical imaging methods make monitoring of the membrane potential feasible. With this technique, one can observe different optical signals depending on optical properties, in response to membrane potential, using voltage-sensitive dyes. We used the multiple-site optical imaging system to investigate membrane potentials of the isolated, cultured spiral ganglion cells (SCGs) from newborn mice. We used a voltage-sensitive absorption dye. With high potassium (150 mM K+) exposure, the absorbency of SGCs stained with voltage-sensitive dye increased temporarily under a 700-nm interference filter at approximately 0.3%. This detection of the depolarization of SGCs using an optical recording technique with a voltage-sensitive dye is important because it shows that the simultaneous measurement of activity in a variety of regions is possible, as is the exploring of the intercellular signalling pathway.

Animals↗
PubMed2000

Crucial role of the membrane potential for ATP synthesis by F(1)F(o) ATP synthases.

ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na(+)). The proton-motive force consists of two components, the transmembrane proton concentration gradient (delta pH) and the membrane potential. The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on delta pH only. Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis. Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium. These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the F(o) motor of Propionigenium modestum ATP synthase.

Adenosine Triphosphate↗
PubMed1999

Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.

The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.

Antibodies↗
PubMed2001

New control of mitochondrial membrane potential and ROS formation--a hypothesis.

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.

Adenosine Triphosphate↗
PubMed1999

Depolarization of osteoclast plasma membrane potential by 17beta-estradiol.

The effect of estrogen on plasma membrane potential of isolated avian osteoclasts was examined through the use of a fluorescent potential-sensitive dye, bis-(1,3-dibutylbarbiturate) trimethine oxonol, also known as bis-oxonol. A decrease in potential was observed within seconds of addition of 17beta-estradiol. Ouabain, a specific Na+K+-ATPase inhibitor, and BaCl2, an inhibitor of the inwardly rectifying K+ channel, blocked the estrogen response. Verapamil and lanthanum chloride (LaCl3), inhibitors of inward Ca2+ channels, and 4'4-diisothiocyanatostilbene-2'2-disulfonic acid (DIDS), an inhibitor of Cl- channels, did not affect the depolarization. Herbimycin A, a tyrosine kinase inhibitor, also had no effect on the decreased membrane potential. These data provide evidence which suggests that estrogen regulates osteoclasts through ion channel activities. The change in K+ channel activity was observed within seconds of addition of 17beta-estradiol, indicating an action at the level of the plasma membrane.

Animals↗
PubMed1986

Regulation of voltage-dependent Ca2+ channels of neuronal cells by chronic changes in membrane potential.

The regulation of voltage-dependent Ca2+ channels by chronic changes in membrane potential was studied by measuring the binding of [3H]nitrendipine to membranes from PC12 pheochromocytoma cells. Depolarization of the cells by elevation of extracellular K+ caused up to a 45% loss of [3H]nitrendipine binding sites within 4 days without any change in Kd. Repolarization of the cells caused a return to control binding within 1-2 days, showing that either decreases or increases in membrane potential can cause reversible changes in [3H]nitrendipine binding sites.

Animals↗
PubMed2003

Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis.

Little is known about the temporal relationship between mitochondrial and plasma membrane potential changes and outer mitochondrial membrane permeabilization during apoptosis. Confocal imaging of breast carcinoma and HeLa cells stably transfected with cytochrome-C-GFP demonstrated that mitochondria rapidly depolarized after the release of the fusion protein into the cytosol. Of note, mitochondria did not completely depolarize but established a new steady-state level that could be further dissipated by treatment with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. Treatment with the F(O)F(1)-ATP-synthase inhibitor oligomycin likewise induced a collapse of this steady-state level, suggesting that F(O)F(1)-ATP-synthase reversal maintained mitochondrial potential after outer mitochondrial membrane permeabilization. Treatment with a broad spectrum caspase inhibitor failed to inhibit the partial depolarization of mitochondria during apoptosis, yet potently abolished the activation of effector caspases detected by fluorescence resonance energy transfer analysis in the same experiment. Interestingly, the onset of mitochondrial depolarization was always coupled with a depolarization of the plasma membrane potential. This was associated with the degradation of the regulatory Na(+)/K(+)-ATPase beta-subunit, and both events were blocked by caspase inhibition. Our results demonstrate that outer mitochondrial membrane permeabilization coordinates the depolarization of both membrane potentials during apoptosis.

Apoptosis↗
PubMed1983

The dibromothymoquinone effect on membrane potential generation in Rhodospirillum rubrum chromatophores.

2,5-Dibromo-3-methyl-6-isopropyl benzoquinone (DBMIB) inhibits the light-dependent membrane potential generation in Rhodospirillum rubrum chromatophores. The inhibition is relieved by electron donors and is obviously due to oxidation of the photosynthetic electron transfer chain components. In addition, high DBMIB concentrations elicit another effect probably caused by disruption of quinone functions in chromatophores. However, in quinone-depleted chromatophores and proteoliposomes containing the P-870 reaction center and light-harvesting antenna complexes, DBMIB stimulates membrane potential generation in the light, probably restoring some of the quinone-dependent processes in the membrane. DBMIB inhibits the inorganic pyrophosphate- and ATP-induced membrane potential generation in chromatophores.

Adenosine Triphosphate↗
PubMed1987

Effect of anisotonic media on volume, ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture.

Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K+, Cl- and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K+ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na+ and Cl- increase and followed by a slow recovery of the initial K+ and Na+ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na+ and Cl- conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K+ and Cl- content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca2+ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl- by nitrate. When cells in a monolayer were incubated in a hypotonic K2SO4 medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K+ and Na+ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca2+ concentration.

Amino Acids↗
PubMed1975

The independence of membrane potential and potassium activation of the sodium pump in muscle.

The membrane potential (Em) of sartorius muscle fibers was made insensitive to [K+] by equilibration in a 95 mM K+, 120 mM Na+ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with Em sensitive to [K+]. In addition, in the presence of 10 mM K+, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10- minus 4 M oubain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.

Animals↗
PubMed1998

Mitochondrial membrane potential supported by exogenous cytochrome c oxidation mimics the early stages of apoptosis.

Mitochondria isolated from rat liver, incubated with inhibitors of respiratory Complexes I and III but in the presence of added NADH as a source of reducing equivalents, generate a membrane potential, the extent of which is comparable to that supported by the oxidation of intramitochondrial respiratory substrates. A catalytic amount of exogenously added cytochrome c is required in order for this membrane potential to be observed. Such an experimental approach mimics the early stages of the apoptotic program of mammalian cells. This has been reported to be characterized by the extrusion of the bulk of intermembrane pool of cytochrome c while the mitochondria continue to preserve their membrane potential. The data obtained are consistent with the proposal that the bi-trans-membrane electron transport pathway, activated by the transfer of cytochrome c from mitochondria to the cytosolic compartment, may be directly involved in the generation of the mitochondrial membrane potential at the beginning of the cell death process.

Animals↗