Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na⁺/K⁺ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium. © 1994 Wiley-Liss, Inc.     

IgE receptor-mediated depolarization of rat basophilic leukemia cells measured with the fluorescent probe bis-oxonol

Receptor-mediated changes in plasma membrane potential were recorded in rat basophilic leukemia (RBL) cells with the potential-sensitive fluorescent indicator bis-oxonol. Depolarization of the mitochondria with metabolic inhibitors was not detected by bis-oxonol, suggesting that only potential changes across the plasma membrane were being measured. The resting membrane potential of RBL cells was largely generated by the equilibrium distribution of K+ and not through electrogenic activity of the sodium pump. Depolarization was maintained as long as IgE receptors remained aggregated. We believe that at physiologic calcium concentrations a large portion of the measured potential change may be due to calcium influx across the plasma membrane. Prevention of calcium influx by lanthanum, disruption of aggregated receptors, or prior depolarization in a high K+ saline solution completely inhibited the antigen-induced depolarization. The time course of the antigen-stimulated increase in bis-oxonol fluorescence was similar, but not identical, to the antigen-stimulated rise in cytoplasmic free ionized calcium measured with fura-2. Antigen-stimulated depolarization was inhibited by removing both calcium and sodium and could be restored by the addition of either ion. Reduction of total cellular adenosine triphosphate inhibited depolarization in response to antigen stimulation.     

Significant differences between procyclic and bloodstream forms of Trypanosoma brucei in the maintenance of their plasma membrane potential

The plasma membrane potential (deltapsi) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of deltapsi in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H(+)-ATPase inhibitors (e.g. dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A1). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium-rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the deltapsi of the procyclic stages in a sodium-rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H(+)-ATPase inhibitor dicyclohexylcarbo-diimide, indicating the presence of an ouabain-sensitive sodium pump whose activity is masked by the H(+)-ATPase. Unlike procyclic forms, the deltapsi of bloodstream-stage trypomastigotes was markedly sensitive to extracellular Na+ and K+ concentrations. Thus, there are significant differences between procyclic and bloodstream forms in the maintenance of the deltapsi and in their permeability to cations.     

Transmembrane potential of isolated rat alveolar type II cells

Type II cells were isolated from rat lungs by elastase digestion and purified by centrifugal elutriation. The fluorescent dye, Di-S-C3(5), was used as a probe to monitor transmembrane potential (Em) of cells suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered medium. With this technique, the Em of type II cells was estimated to be -27 +/- 2 mV. This resting Em is very close to the equilibrium potential for chloride (-21 mV), which suggests that chloride is passively distributed in type II cells. The resting Em of type II cells is more dependent on the extracellular concentration of potassium (K+) than on external sodium (Na+); i.e., the membrane depolarizes as external sodium is replaced by potassium, suggesting that in unstimulated type II cells the membrane is more permeable to potassium than to sodium. In addition, the resting potential appears to be due, in part, to the activity of a ouabain-sensitive, Na-K pump, which acts to hyperpolarize type II cells. Addition of a membrane perturbant, phorbol myristate acetate (PMA, 10 micrograms/ml), to a type II cell suspension results in an increase in oxygen consumption and membrane depolarization. Both of these responses are sodium dependent and thus appear to be linked to a PMA-induced increase in sodium permeability.     

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.     

Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.     

Effect of the Ca ionophore A-23187 on the plasmatic and mitochondrial potentials of the brain synaptosomes in rats: fluorescence measurements

The applicability of the potential-sensitive dye diS-C3-(5) for the study of A23187 + Ca2+ induced plasma membrane hyperpolarization was tested in rat brain synaptosomes. An appropriate dye synaptosome ratio was chosen for the fluorescence titration dye in Ca-free Krebs-Ringer solution. The fluorescence intensity of the probe was increased upon the addition of Ca2+ (1 microM) to the synaptosomes in the presence of A23187 (1 microM). The effect of Ca2+ + A23187 persisted in a Na+-free medium or when Na+ channels were inhibited by tetrodotoxin as well as in high K+-depolarized synaptosomes (75 microM KCl). In the presence of oligomycin or a protonophore (1 microM) the effect of Ca2+ + A23187 was suppressed. This suggests that the A23187-induced fluorescence increase is due to a depolarization of intrasynaptosomal mitochondria. Therefore, the use of the dye diS-C3-(5) for the study of Ca-induced hyperpolarization does not seem to be feasible unless a quantitative model of changes in fluorescence related to the plasma and mitochondrial membrane potentials is elaborated.     

Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the psi m or the psi pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.     

Spectroscopic investigations of the potential-sensitive membrane probe RH421.

The absorbance spectra, fluorescence emission and excitation spectra, and fluorescence anisotropy of the potential-sensitive styryl dye RH421 have been investigated in aqueous solution and bound to the lipid membrane. The potential-sensitive response of the dye has been studied using a preparation of membrane fragments containing a high density of Na+, K(+)-ATPase molecules. In aqueous solution the dye is sensitive both to changes in pH and ionic strength. Evidence has been found that the dye readily aggregates in aqueous solution. Aggregation is enhanced by an increase in ionic strength. The aggregates formed display a low fluorescence intensity. At high pH values (above approx. 8) changes in the dye's fluorescence spectra are observed, which may be due to a reaction of the dye with hydroxide ions. When bound to the membrane the dye also exhibits concentration-dependent fluorescence changes. The potential-sensitive response of the dye in Na(+),K(+)-ATPase membrane fragments after addition of MgATP in the presence of Na+ ions cannot be explained by a purely electrochromic mechanism. The results are consistent with either a potential-dependent equilibrium between membrane-bound dye monomers and membrane-bound dimers, similar to that previously proposed for the dye merocyanine 540, or with a field-induced structural change of the membrane.     

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.     

Spectral imaging of MC540 during murine and human colon carcinoma cell differentiation.

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147-153, 2001)     

Regulation of the Na(+)-K+ pump activity and estimation of the reserve capacity in intact rat peritoneal mast cells

Evidence is provided that regulation of the Na(+)-K+ pump activity in rat peritoneal mast cells occurs mainly through stimulation of the pump from inside the plasma membrane by sodium. It is demonstrated that there is a large reserve capacity for the exchange of intracellular sodium with extracellular potassium in these cells. The maximal pump activity was estimated to be 3230 pmol/10(6) cells per min and Km for extracellular potassium was 1.5 mM.     

A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

Membrane potential induced by external electric field pulses can be followed with a potentiometric dye.

A potential-sensitive dye was recently used to measure the spatial variation in the membrane potential induced by an externally applied electric field. In this work, we demonstrate that the time course of these induced potentials can also be followed. Two experimental systems were explored. Dye fluorescence from HeLa cells could be modulated by a train of field pulses; the relative fluorescence change measured with a lock-in amplifier was linear with the field and similar to the fluorescence responses obtained in the static measurements. A model membrane system consisting of a hemispherical bilayer allowed convenient measurement of the dye absorbance change as a function of the bathing solution conductivity. The charging time of the membrane was inversely related to the aqueous conductance as predicted by the theoretical solution to Laplace's equation.     

Fluorimetric determination of the resting potential changes associated with the chemotactic response in Paramecium

1. (1) Evidence is presented which indicates that the carbocyanine dye (3,3′ dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across the plasma membrane of the ciliated protozoan Paramecium.

2. (2) The dye at low concentrations ( 1 μM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response.

3. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration.

4. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential.

5. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods.

6. (6) Both cations which induce a negative chemotactic response in Paramecium (K⁺, Na⁺, Ba²⁺) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.

Monitoring of real changes of plasma membrane potential by diS-C3(3) fluorescence in yeast cell suspensions

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.     

Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

Summary Changes in the fluorescence intensity of the dye 3-3 dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10^–9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg²⁺ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.Presented in part at the 17th Annual Cell Biology Meeting.Cell Biol. 75:103a, 1977.     

Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?

The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.     

The use of a cyanine dye in measuring membrane potential in yeast

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.