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Quantifying the effect of membrane potential in chemical osmosis across bentonite membranes by virtual short-circuiting.

Clay liners are charged membranes and show semipermeable behavior regarding the flow of fluids, electrical charge, chemicals and heat. At zero gradients of temperature and hydrostatic pressure, a salt concentration gradient across a compacted clay sample induces not only an osmotic flux of water and diffusion of salt across the membrane but also an electrical potential gradient, defined as membrane potential. Laboratory experiments were performed on commercially available bentonite samples in a rigid-wall permeameter connected to two electrically insulated fluid reservoirs filled with NaCl solutions of different concentrations and equipped with Ag/AgCl electrodes to measure the electrical potential gradient. The effect of membrane potential could be cancelled out by short-circuiting the clay with the so-called virtual shortcut. The potential gradient across the sample is brought to zero with a negative feedback circuit. It was observed that the water flux and the diffusion of Cl- were hindered by the occurrence of a membrane potential, indicating that an electroosmotic counterflow is induced. Flow parameters were calculated with modified coupled flow equations of irreversible thermodynamics. They were in excellent agreement with values reported in the literature. Comparing the method of short-circuiting with a study elsewhere, where the electrodes were physically short-circuited, it was shown that the virtual shortcut is more appropriate because physically short-circuiting induces additional effects that are attributed to the fluxes.

Journal Article↗

The relationship of afterhyperpolarizations of extraocular motoneurons to membrane potential.

The effect of membrane potential displacements caused by external current injection on the amplitudes of two antidromically evoked afterhyperpolarizations (AHP1 and AHP2) was measured in extraocular motoneurons. AHP1 had a reversal potential of-70mV and an average compensation for displacements from the reversal potential ("compensation gain") of -0.36. AHP2 had a reversal potential of-80mV and a compensation gain of -0.08. Measurements of input resistance of these motoneurons demonstrate a 30-50% decrease at depolarized membrane potentials. This rectification could account for the apparent lack of summation of AHP2. The functional role of AHP1 and AHP2 in the regulation of discharge frequencies of extraocular motoneurons during nromal eye movements is discussed.

Animals↗

Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry.

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388D1 and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (E(s)) frequently reported. Upon microelectrode entry into the cell (P388D1), the recorded potential drops to a peak value (E(p)) (mean -37 mV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (E(n)) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (E(s)) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (E(h)) is -30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U.V., A.M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288 for other change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of E(p), E(n), E(h), E(s), and membrane resistance (R(m)) measured for the other macrophages were similar to those of P388D1. From these results we conclude that E(p) is a better estimate of the true membrane potential of macrophages than E(s), and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes.

Animals↗

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in a Ca2+-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3'-diethyl-2,2'-thiadicarbocyanine (Di-S-C2(5] to monitor inside positive or inside negative membrane potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca2+-ATPase, while negative potential disrupts preformed Ca2+-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca2+-ATPase.

Animals↗

Role of ions and membrane potential in uptake of serotonin into plasma membrane vesicles from mouse brain.

Plasma membrane vesicle preparations from mouse cerebral cortex actively accumulated [3H]serotonin upon the imposition of a K+ gradient (in greater than out), a Na+ gradient (out greater than in), and the presence of external Cl-. Maximal stimulation of uptake by internal K+ occurred at 15 mM and half-maximal stimulation at 2 mM. Internal K+ did not enhance uptake merely via generation of a membrane potential because simultaneous parallel increases in internal and external K+ concentration also stimulated uptake. External Cl- increased serotonin uptake with a Km of 18 mM and a Hill number of 1.0, suggesting a requirement for one chloride ion for transport. Uptake could not be driven by internal H+ instead of K+. Estimation of the membrane potential by the distribution of triphenylmethylphosphonium ion showed a modest effect of valinomycin (1-20 microM) in increasing the potential from -19 to -31 mV accompanied by an increase in serotonin uptake. Proton ionophores prevented this effect of valinomycin and, by themselves, reduced the potential to -6 mV, but did not affect serotonin transport. A model is proposed for serotonin transport in brain plasma membrane vesicles that is similar to the model for porcine blood platelet vesicles as far as electroneutrality and stimulation by K+, Na+, and Cl- are concerned, but that is different in substitution of internal H+ for K+.

Animals↗

A quantitative assessment of the use of 36Cl- distribution to measure plasma membrane potential in isolated hepatocytes.

The plasma membrane potential of isolated rat hepatocytes was clamped at different values between 0 and -68 mV by addition of valinomycin in the presence of different extracellular concentrations of K+, and measured by the distribution of 86Rb+ between cells and medium. 36Cl- distribution came to steady state in 10-15 min. This steady-state distribution was compared to the plasma membrane potential over a range of values. 36Cl- distribution provided an accurate measurement of plasma membrane potential between -4 and -40 mV. At higher potentials intracellular chloride concentration is less than 20% of the extracellular concentration and errors due to uncertainties in the measurement of intracellular volume and of the contamination of cell pellets by extracellular medium precluded accurate determination of membrane potential: thus in our experiments 36Cl- underestimated the plasma membrane potential at -68 mV by 8 mV.

Adenosine Triphosphate↗

Possible energization of K+ accumulation into metabolizing yeast by the protonmotive force. Binding correction to be applied in the calculation of the yeast membrane potential from tetraphenylphosphonium distribution.

Membrane potentials of yeast cells, Saccharomyces cerevisiae, calculated from the equilibrium distribution of tetraphenylphosphonium (TPP) between cell-water and medium should be corrected for a contribution due to binding of TPP to intracellular constituents. The magnitude of this correction depends upon the way in which it is determined. In cells permeabilized by boiling, cell-binding is much higher than in cells permeabilized by repeated freezing and thawing. The binding corrections are 75 +/- 1 mV and 49 +/- 7 mV, respectively. The binding correction obtained from TPP distribution between deenergized cells and medium is much lower and amounts to 19 +/- 9 mV. The latter value is probably more reliable. It is supposed that permeabilization of the cells by boiling or repeated freezing and thawing unmasks potential TPP binding groups in the cell. The K+ accumulation into anaerobically metabolizing yeast cells can be accounted for almost quantitatively by a cotransport of protons and K+ ions if the lower binding correction is applied. This means that K+ accumulation into the yeast cell may be driven by the sum of the protonmotive force and the membrane potential.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗

Evidence for the role of non-quantal acetylcholine in the maintenance of the membrane potential of rat skeletal muscle.

1. Resting membrane potentials of rat diaphragm muscles cultured in Trowell T8 medium were measured in vitro. After 3 hr in culture the resting membrane potential of muscle fibres within 2.5 mm of nerve section (;near') was -68.3 +/- 0.4 mV (nineteen preparations). This was significantly lower (P < 0.001) than the resting potential (-74.0 +/- 0.4 mV) measured in muscle fibres 8-10 mm from the site of nerve section (;far') in the same preparations. A difference between the ;near' and the ;far' fibres was maintained in muscles cultured for 6 and 12 hr. Miniature end-plate potentials were present in both ;near' and ;far' fibres cultured for 3 and 6 hr and ceased after 12-15 hr.2. The presence of carbamylcholine (10(-7) or 10(-8) M) maintained the resting membrane potential of ;near' fibres close to that of ;far' fibres at 3, 6 and 12 hr. For example, at 3 hr in the presence of 10(-8) M-carbamylcholine the mean resting potential was 75.6 +/- 0.5 mV in ;near' fibres and 76.1 +/- 0.4 mV in ;far' fibres (four preparations). A similar effect was produced in preparations exposed to anticholinesterases: diisopropylphosphorofluoridate (DFP) (10(-7) M), neostigmine (10(-7) M) or physostigmine (10(-5) M).3. Agents that blocked acetylcholine receptors had the reverse effect. In the presence of alpha-bungarotoxin (1 mug/ml.) or d-tubocurarine (10(-5) M) the resting membrane potential of ;far' fibres was reduced to the level of ;near' fibres over the 24 hr period of observation. For example, at 3 hr in the presence of alpha-bungarotoxin the mean resting potential was 67.2 +/- 0.5 mV in ;near' fibres and 68.5 +/- 0.6 mV in ;far' fibres (six preparations). The effect of d-tubocurarine was reversible.4. When muscles were cultured in Ca(2+)-free medium containing 1 mM-EGTA and 10 mM-Mg(2+), there was no difference in membrane potential between ;near' and ;far' fibres and physostigmine (10(-5) M) was ineffective in raising the membrane potential of ;near' fibres.5. It is suggested that non-quantal acetylcholine released from nerve terminals maintains the membrane potential of muscle fibres through a Ca(2+)-dependent mechanism.

Acetylcholine↗

Stimulus dependence of two-state fluctuations of membrane potential in cat visual cortex.

Membrane potentials of cortical neurons fluctuate between a hyperpolarized ('down') state and a depolarized ('up') state which may be separated by up to 30 mV, reflecting rapid but infrequent transitions between two patterns of synaptic input. Here we show that such fluctuations may contribute to representation of visual stimuli by cortical cells. In complex cells of anesthetized cats, where such fluctuations are most prominent, prolonged visual stimulation increased the probability of the up state. This probability increase was related to stimulus strength: its dependence on stimulus orientation and contrast matched each cell's averaged membrane potential. Thus large fluctuations in membrane potential are not simply noise on which visual responses are superimposed, but may provide a substrate for encoding sensory information.

Anesthesia↗

Association between cell membrane potential and breast cancer.

Cell membrane potentials were measured in breast tissue and in breast epithelial cells to explore the relation between cell membrane potentials, oncogenesis and electrical potentials previously measured on the surface of the breast. The mean membrane potential in breast biopsy tissue from 9 women with infiltrating ductal carcinoma was significantly depolarized, compared with values measured in tissue from 8 women with benign breast disease. Depolarization was also observed in transformed breast epithelial cells, compared with normal breast cells; the transformed cells were particularly sensitive to the action of K+ channel blockers. The results were consistent with previous observations of electropositivity of skin sites over malignant tumors of the breast.

Adult↗

Changes of the membrane potential in striatal synaptoneurosome, synaptosome and membrane sac preparations induced by glutamate, kainate and aspartate as measured with a cyanine dye DiS-C2-(5).

The effects of glutamate, kainate and aspartate on the membrane potential of striatal synaptoneurosome, synaptosome and membrane sac preparations were studied by using a potential sensitive cyanine dye DiS-C2-(5). Excitatory amino acids glutamate and aspartate had a depolarizing effect on synaptoneurosomes. 7.9 microM glutamate and 2.8 microM aspartate produced a half-maximal response. Depolarizations induced by glutamate and aspartate were dependent on the concentration of extracellular sodium ions, a maximal response occurred at around 40 mM of external Na+. Kainate induced a dual effect on synaptoneurosomes. In a standard Na+-based medium a hyperpolarization, likely due to inhibition of a presynaptic sodium-dependent glutamate uptake, predominated over a postsynaptic kainate receptor-mediated depolarization that was observed when electrogenic glutamate uptake was inhibited. This interpretation was supported by results obtained with synaptosome and membrane sac preparations. In a standard Na+-based medium kainate had a hyperpolarizing effect on synaptosomes while in the membrane sac preparation kainate induced a depolarization.

Animals↗

The production of reactive oxygen species in intact isolated nerve terminals is independent of the mitochondrial membrane potential.

Dependence on mitochondrial membrane potential (deltapsim) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H2O2 generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250 nM) or DNP (50 microM) and then the rate of H2O2 production was followed. Neither of the uncouplers had a significant effect on the rate of H2O2 production induced by rotenone or antimycin. Inhibition of the F0F1-ATPase by oligomycin, which also eliminates deltapsim in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H2O2 production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of deltapsim. In addition, we detected a significant antimycin-induced H2O2 production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.

Animals↗

Basolateral membrane potential of a tight epithelium: ionic diffusion and electrogenic pumps.

The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K+) = 72 mM and (Cl-) = 15.8 mM. Cl- but not K+ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (PNa/PK) was 0.044, and the chloride to potassium permeability ratio (PCl/PK) was 1.17. Since K+ was not in electrochemical equilibrium, intracellular (K+) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K+and C1- the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20mV (cell interior negative). This potential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na+. An equivalent electrical circuit for Na+ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.

Animals↗

The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells.

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na(+) concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = -0.004 Em + 0. 03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 microM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25-100 microM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 microM) and oligomycin (4 microM) did not influence F. I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.

Animals↗

[Effect of mitochondrial membrane potential in the neurotoxicity of artemether].

OBJECTIVE: To study the effect of mitochondrial membrane potential and cellular membrane permeability in the neurotoxicity of artemether. METHODS: Mitochondrial membrane potential and cellular membrane permeability in pheochromocytoma cell and primary cultured rat neuronal cell were measured by flow cytometry, using rhodamine 123(Rh123) and PI as fluorescent dye, respectively. Mitochondrial swelling was measured by spectrometer. RESULTS: Artemether could decrease the mitochondrial membrane potential and increase the cellular membrane permeability in both cell types in a dose-dependent manner. In addition, artemether led to mitochondrial swelling with dose-effect and time-effect relationships. CONCLUSION: The decrease of mitochondrial membrane potential plays an important role in the neurotoxicity of artemether. The drug can change the mitochondrial membrane potential and mitochondrial swelling by affecting the permeability transition pore complex located in the mitochondrial membrane, increasing cellular membrane permeability, causing an obstruction and finally leading to neurotoxicity.

Animals↗

Pentoxifylline improves resting membrane potential in sepsis.

The resting membrane potential (RMP) of skeletal muscle cells was investigated in a hyperdynamic animal model of sepsis. Reduced membrane potential is known to parallel the clinical course of sepsis and hemorrhagic shock in animal models. Pentoxifylline (PTF), a dimethyl xanthine derivative of the theophylline family, may improve the metabolic effects of sepsis. A cecal ligation and puncture (CL&P) sepsis model in Wistar rats was used in which RMP was measured with differing administration regimes of PTF. The RMP decreased less rapidly in our study when PTF was administered after CL&P and this effect was most noticeable after 18 hours. Administration of PTF before CL&P had little effect on membrane potential fall-off. These results suggest a potential role for PTF in the management of sepsis.

Animals↗