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PubMed1987

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential.

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3'-dipentyloxacarbocyanine (di-O-C5(3)) or 3,3'-dipropylthiadicarbocyanine (di-S-C3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 microgram/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP+) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P less than 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P less than 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.

Animals↗
PubMed1987

Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis.

Lipophilic ions are widely used as probes for measuring membrane potentials. Since binding of the probes to the membrane interferes with the accurate estimation of the membrane potential, it is necessary to clarify the characteristics of probe binding to membranes. The present paper deals with the binding of lipophilic cations to liposomes. The results can be summarized as follows: (1) The binding of triphenylmethylphosphonium, its homologues and tetraphenylphosphonium to liposomes of dipalmitoylphosphatidylcholine followed the Langmuir adsorption isotherm. (2) Spin-labeled lipophilic cations were synthesized and the binding to liposomes of egg phosphatidylcholine was examined. The binding also followed the Langmuir adsorption isotherm. The dissociation constant (the concentration giving half-maximal binding), K, was independent of the temperature, indicating that the binding is entropy-driven. (3) The binding was influenced by the fluidity of the membrane. Except in the case of triphenylmethylphosphonium (TPMP+), K and A (maximum amounts of binding) increased above the transition temperature. In other words, above the phase transition temperature the binding affinity is decreased, while maximum amounts of binding are increased for all phosphoniums used except TPMP+.

Adsorption↗
PubMed1988

Depolarization-induced release of glycine and beta-alanine from plasma membrane vesicles derived from rat brain synaptosomes.

Glycine and beta-alanine actively loaded into brain synaptic plasma membrane vesicles were released into the external medium by using the classical depolarization agents high K+ and veratridine. This release occurs via a Ca2+-independent process. Measurements of membrane depolarization using tetraphenylphosphonium uptake show a close correlation between changes in the membrane potential and stimulation of the efflux process. Results shown herein and previously reported by our group (Aragón, M.C. and Giménez, C. (1986) Biochim. Biophys. Acta 855, 257-264; Agulló, L., Jiménez, B., Aragón, M.C. and Giménez, C. (1986) Eur. J. Biochem. 159, 611-617), suggest that the glycine and beta-alanine transport systems in synaptic plasma membranes are susceptible of modulation by changes in ionic fluxes and hence in the membrane potential, similar to those occurring during depolarization and repolarization.

Action Potentials↗
PubMed1988

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer.

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.

Algorithms↗
PubMed1989

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↗
PubMed1991

Resting membrane potential in 41A3 mouse neuroblastoma cells. Effect of increased glucose and galactose concentrations.

Neuroblastoma cells were used to examine the effect of high concentrations of glucose or galactose and accumulation of polyols on the resting membrane potential. Polyol levels are increased and myo-inositol content decreased when neuroblastoma cells are chronically exposed to media containing 30 mM glucose or 30 mM galactose compared to cells grown in media containing 30 mM fructose. Furthermore, the 6 h accumulation and incorporation into phospholipid of extracellular myo-inositol is decreased in cells exposed to media containing 30 mM glucose or 30 mM galactose compared to cells grown in media containing 30 mM fructose. The resting membrane potential was determined by examining the steady-state accumulation of the lipophilic cation tetra[3H]phenylphosphonium bromide (TPP+). The resting membrane potential of cells grown in media containing 30 mM fructose is about -70 mV which is very similar to the resting membrane potential of cells grown in unsupplemented media. The resting membrane potential is significantly decreased in cells grown in media containing 30 mM glucose or 30 mM galactose. myo-Inositol metabolism and content and polyol levels are maintained at near normal values and the resting membrane potential is improved when media containing 30 mM glucose or 30 mM galactose are supplemented with 0.4 mM sorbinil. Acute exposure of neuroblastoma cells to 2 mM ouabain had no significant effect on [3H]TPP+ accumulation. This suggests that acute inhibition of Na+/K+ pump activity does not decrease the resting membrane potential of neuroblastoma cells. The decrease in resting membrane potential may be induced by the metabolic abnormalities and/or chronic decrease in Na+/K+ pump activity which occur when neuroblastoma cells are chronically exposed to increased glucose or galactose concentrations.

Animals↗
PubMed1991

Monensin-mediated antiport of Na+ and H+ across liposome membrane.

The mechanism of monensin-mediated transport of Na+ and H+ across large unilamellar liposome membrane was investigated. The inside negative membrane potential (delta psi) was generated by the addition of monensin to the liposomes with an outward Na+ gradient. The effects of intravesicular H+ bufferring power and medium pH on the initial rates of delta psi formation, Na+ efflux and H+ influx were examined. The results showed that (i) the initial Na+ flux (JNa) was larger than the initial H+ flux (JH) at any H+ bufferring power, (ii) the JH increased with increasing inner buffer concentration, but the effect of H+ bufferring power on the JNa was small, (iii) the initial rate of delta psi formation increased linearly with the increase in the value of (JNa-JH), and (iv) the JNa increased with increasing H+ concentration. The generation of delta psi was not due to H+ leak from the liposome, since the delta psi was generated even when H+ concentration gradient was inwardly directed. The monensin-mediated transport of Na+ and H+ in this system occurred at the ratio of Na+/H+ greater than 1.0 and the resultant net electric charge efflux is the cause of the inside negative membrane potential. Tetraphenylphosphonium retarded both the delta psi formation and the H+ influx, but did not affect the Na+ efflux, suggesting that the driving force of H+ influx is the inside negative membrane potential generated by Na+ efflux. This idea also well accounts for the observed H+ bufferring power effects on the Na+ efflux, H+ influx and delta psi formation. It was suggested that Na+ was transported in the form of 1:1 complex between protonated monensin and Na+.

Arylsulfonates↗
PubMed1992

Measurement of plasma membrane potential in isolated rat hepatocytes using the lipophilic cation, tetraphenylphosphonium: correction of probe intracellular binding and mitochondrial accumulation.

The lipophilic cation tetraphenylphosphonium (TPP+) has been extensively utilized as the probe for the membrane potential (Vm) in various cells. For application to mammalian cells, however, two serious problems require resolution: (1), correction of TPP+ binding to intracellular constituents and (2), estimation of the considerable TPP+ accumulation in mitochondria. We propose here a simple corrective method for the TPP+ binding and its accumulation. TPP+ distribution is assumed as: (1), two compartments (a cytosolic and a mitochondrial space); (2), a proportional relationship between TPP+ bound amount and its unbound concentration in each compartment. We theoretically derived the simple equation: Vm = - RT/F ln(C/Mphys ratio/C/Mabol ratio) where R, T and F have their usual thermodynamic significance. Here, the C/M ratio is defined as the ratio of TPP+ concentration of apparent intracellular to extracellular space. The suffixes phys and abol, respectively, mean the physiological and solely Vm-abolished conditions. This equation was checked with hepatocytes, because estimating hepatocytes Vm with TPP+ distribution is not considered possible because of the relatively high mitochondrial content. The selective Vm abolition was achieved by permeabilization with 20 microM of amphotericin B. The Vm value was, thus, estimated to be -38.6 +/- 0.3 mV, compatible with those obtained with microelectrodes in other laboratories. Vm in hepatocytes is composed of transmembrane K+ diffusion potential (-20.6 +/- 0.3 mV) and electrogenic Na+/K(+)-ATPase (-19.6 +/- 0.4 mV). Addition of rheogenic L-alanine caused a transient but significant depolarization (from control to -34 +/- 0.3 mV). These results taken together indicate that hepatocyte Vm can be accurately determined with the present simple method, so that it may possibly be applicable to the evaluation of Vm in other mammalian cells.

Amphotericin B↗
PubMed1993

The membrane potential has no detectable effect on the phosphocholine headgroup conformation in large unilamellar phosphatidylcholine vesicles as determined by 2H-NMR.

In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the alpha- or beta-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K(+)-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to delta psi, which reorients the phosphocholine headgroups in the opposite bilayer-water interfaces.

Benzothiazoles↗
PubMed1994

Small lipid-soluble cations are not membrane voltage probes for Neurospora or Saccharomyces.

Small lipid-soluble cations, such as tetraphenylphosphonium (TPP+) and tetraphenylarsonium (TPA+) are frequently used as probes of membrane voltage (delta psi, or Vm) for small animal cells, organelles, and vesicles. Because much controversy has accompanied corresponding measurements on 'walled' eukaryotic cells (plants, fungi), we studied their transport and relation to Vm in the large-celled fungus Neurospora crassa-where Vm can readily be determined with microelectrodes-as well as in the most commonly used model eukaryotic cell, the yeast Saccharomyces cerevisiae. We found no reasonable conditions under which the distribution of TPP+ or TPA+, between the cytoplasm (i) and extracellular solution (o), can serve to estimate Vm, even roughly, in either of these organisms. When applied at probe concentrations (i.e., < or = 100 microM, which did not depolarize the cells nor deplete ATP), TPP+ stabilized at ratios (i/o) below 30 in both organisms. That would imply apparent Vm values positive to -90 mV, in the face of directly measured Vm values (in Neurospora) negative to -180 mV. When applied at moderate or high concentrations (1-30 mM), TPP+ and TPA+ induced several phases of depolarization and changes of membrane resistance (Rm), as well as depletion of cytoplasmic energy stores. Only the first phase depolarization, occurring within the perfusion-turnover time and accompanied by a nearly proportionate decline of Rm, could have resulted from TPP+ or TPA+ currents per se. And the implied currents were small. Repeated testing, furthermore, greatly reduced the depolarizing effects of these lipid-soluble ions, implicating an active cellular response to decrease membrane permeability.

Adenosine Triphosphate↗
PubMed1994

Cation effluxes associated with the uptake of TPP+, TPA+, and TPMP+ by Neurospora: evidence for a predominantly electroneutral influx process.

Previously observed anomalies in the transport of lipid-soluble cations (LSI's) - presumed voltage-probe ions-by intact fungal cells [1] prompted a systematic investigation of ion exchanges induced by high (millimolar) concentrations of the particular species tetraphenylphosphonium ion (TPP+), tetraphenylarsonium ion (TPA+), and triphenylmethylphosphonium ion (TPMP+). With low extracellular free Ca2+ (no calcium added to the medium), influx of the LSI's was biphasic, indicating rapid entry into the cytoplasm followed by sequestration into a subcompartment. The latter process, especially, was strongly inhibited by extracellular Ca2+ (1 mM). Contrary to the expectation for electrophoretically driven entry of LSI's into fungal cells, no major efflux of protons (acidification of the medium) could be measured; in fact, significant alkalinization of the medium was observed. The major cellular inorganic cations, K+ or Na+ (under different conditions), were released during LSI uptake, but with kinetic behavior which clearly ruled out direct coupling to the uptake of TPP+, TPA+, or TPMP+. The major mechanism for entry of these lipid-soluble cations into Neurospora appears to be electroneutral diffusion in combination with one or more hydrophilic anions. Subsequent penetration of the fungal vacuoles would result in binding of LSI's to storage polyanions (viz., polyphosphate) and concomitant displacement of the normal vacuolar cations, such as basic amino acids and polyamines, thus leading to alkalinization of the extracellular medium. The observed effluxes of cytoplasmic K+ and Na+ should result independently from energetic changes (i.e., uncoupling of the mitochondrial) and are most easily described by simple, but asynchronous, changes in the average rate constants for entry and exit of the alkali-metal cations.

Arsenicals↗
PubMed1995

Hydrophobic ion interaction on Na+ activation and dephosphorylation of reconstituted Na+,K(+)-ATPase.

In liposomes with reconstituted shark Na+,K(+)-ATPase an uncoupled Na(+)-efflux and a Na+/Na+ exchange can be induced on inside-out oriented pumps by the addition of external (cytoplasmic) Na+ and MgATP to liposomes that either do not contain Na+ (and other alkali cations), or include 130 mM Na+ internally (extracellular). Both modes of exchange are electrogenic and accompanied by a net hydrolysis of ATP. The coupling ratio of positive net charges translocated per ATP split is found to be close to 3:1 and 1:1, respectively, for the two modes of exchange reactions at pH 7.0. By addition of the hydrophobic anion tetraphenylboron (TPB-), which imposes a negative electrostatic membrane potential inside the lipid bilayer, the ATP hydrolysis accompanying uncoupled Na+ efflux is increased with increasing TPB- concentrations. Cholesterol which increases the inner positive dipole potential of the bilayer counteracted this activation by TPB- of uncoupled Na+ efflux. Using the structural analog tetraphenylphosphonium (TPP+), which elicits an inside positive membrane potential, ATP hydrolysis accompanying uncoupled Na(+)-efflux is decreased. The rate of dephosphorylation in the absence of extracellular alkali cations was affected in a similar manner, whereas the dephosphorylation in the presence of extracellular Na+ inducing Na+/Na+ exchange was unaffected by the hydrophobic ions. In both modes of exchange the phosphorylation reaction was independent of the presence of hydrophobic ions. The hydrophobic ions affected the apparent affinity for cytoplasmic Na+, indicating that binding of cytoplasmic Na+ may involve the migration of cations to binding sites through a shallow cytoplasmic access channel. The results are in accordance with the simple electrostatic model for charge translocation in which two negative charges in the cytoplasmic binding domain of the Na+,K(+)-ATPase co-migrate during cation transport.

Adenosine Triphosphate↗
PubMed1996

The measurement of transmembrane electrical potential with lipophilic cations.

The binding of lipophilic cation probes of membrane potential to cells was re-examined. Even concentrations of probe molecules as low as 100 nM were found to reduce delta psi and thus many commonly used techniques for delta psi determination are inappropriate. Binding was found to be a linear function of probe concentration and independent of pH. The proportionality constant for binding has been equated to an "apparent binding volume' for [3H]TPP+ with units of microliter/mg dry weight of cells. This "apparent binding volume' is thermodynamically equivalent to the volume of cell membrane multiplied by the partition coefficient of [3H]TPP+ for cell membrane and was equivalent to 9.10 +/- 0.33 microliters/mg dry weight in Enterococcus faecalis. It was concluded that the most accurate method for delta psi determination was to use nanomolar concentrations of lipophilic cations and appropriate correction for energy dependent binding.

Cations↗
PubMed1983

Oxygen-induced inhibition of light-dependent uptake of tetraphenylphosphonium ions as a probe of a direct interaction between photosynthetic and respiratory components in cells of Rhodopseudomonas capsulata.

Light-generated and oxygen-dependent membrane potentials by heterotrophycally grown cells of Rhodopseudomonas capsulata have been investigated by using a tetraphenylphosphonium ion-selective electrode. The results show that respiratory electron transport affects the magnitude of photogenerated membrane potential while photosynthesis seems to either inhibit or stimulate respiration in coupled or uncoupled cells, respectively. These observations have been considered as evidence that the intracytoplasmic membrane system of R. capsulata contain respiratory and photosynthetic apparatuses which are strictly intermingled.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗