valinomycin and tetraphenylphosphonium

The mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoK(ATP) opening that should be observable at the level of the mitochondrion. MitoK(ATP) opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoK(ATP) opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoK(ATP) openers and blockers with those of valinomycin, we show that four independent assays of mitoK(ATP) activity yield quantitatively identical results for mitoK(ATP)-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoK(ATP) opening for mitochondria.

Topics: Adenosine Triphosphate; Animals; Benzofurans; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Decanoic Acids; Diazoxide; Ethers, Cyclic; Hydrogen-Ion Concentration; Hydroxy Acids; Light; Male; Mitochondria, Heart; Mitochondrial Swelling; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Potassium; Potassium Channels; Rats; Rats, Sprague-Dawley; Scattering, Radiation; Uncoupling Agents; Valinomycin

The uptake by light-grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO(3)(2-)) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and by the K(+)/H(+) exchanger nigericin. Notably, the light-driven membrane potential (Delta psi) is enhanced by K(2)TeO(3)> or =200 microM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N-ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a Delta pH-dependent, non-electrogenic process which is likely to involve the phosphate transporter (PiT family).

Topics: Biological Transport; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Electrochemistry; Ethylmaleimide; Kinetics; Light; Membrane Potentials; Nigericin; Onium Compounds; Organophosphorus Compounds; Oxygen; Proton-Motive Force; Rhodobacter capsulatus; Tellurium; Valinomycin

The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (DeltaPsipm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to DeltaPsipm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (DeltaPsimit) induced by valinomycin at low [K+]o, but was reduced by approximately 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (<=15 min) collapsed both DeltaPsipm and DeltaPsimit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 microM) co-operatively depressed -3H-TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N, N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion -Ki 4.0+/-0.9 (S.D.) microM-, while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on DeltaPsipm, but not DeltaPsimit. This report also provides first evidence for a high-affinity amiloride-binding site on the putrescine carrier, which provides new insight into the biochemical properties of this transporter.

Topics: Amiloride; Animals; Binding Sites; Biological Transport; Breast Neoplasms; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Female; Humans; Indicators and Reagents; Kinetics; Mammals; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Putrescine; Sodium-Hydrogen Exchangers; Spermidine; Tumor Cells, Cultured; Valinomycin

System y+ accounts for the majority of L-arginine transport by pulmonary artery endothelial cells (PAEC). Given that membrane potential is a driving force for transport via system y+, we examined the hypothesis that hypoxia inhibits this transport by decreasing membrane potential. Porcine PAEC or plasma membrane vesicles derived from these cells were exposed to normoxia (room air-5% CO2) or hypoxia (0% O2-95% N2-5% CO2). After exposure, L-[3H]arginine transport and/or accumulation of the lipophilic cation [3H]tetraphenylphosphonium, a quantitative sensor of changes in cell membrane potential, were measured. Hypoxia caused reversible time-dependent decrease in L-arginine transport and membrane potential in PAEC and in plasma membrane vesicles. Comparable decreases in membrane potential and L-arginine transport by PAEC were also observed after depolarization induced by KCl or ouabain. Hyperpolarization, induced by valinomycin, increased membrane potential and L-arginine transport in PAEC and plasma membrane vesicles. Valinomycin also prevented the hypoxia-mediated decreases in membrane potential and L-arginine transport in PAEC. These results indicate that hypoxia-induced plasma membrane depolarization is responsible for reduced L-arginine transport by system y+ in hypoxic porcine PAEC.

Topics: Animals; Arginine; Biological Transport; Cell Membrane; Endothelium, Vascular; Hypoxia; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Ouabain; Potassium; Pulmonary Circulation; Sodium; Swine; Valinomycin

Synaptic plasma membrane (SPM) vesicles represent a membrane fraction very useful in studying non-vesicular neurotransmitter release. The procedure described here to isolate SPM vesicles from a crude synaptosomal fraction of sheep brain cortex is quick, simple (ultracentrifugation in a discontinuous density gradient of dextran T110), and combines a high yield (130 micrograms/g brain) with a satisfactory grade of purification. The preparation of SPM vesicles consists of vesicles (approximately 0.54 +/- 0.8 micron diameter) delimited by a single membrane with the native orientation. We are able to ascertain these characteristics on the basis of morphology studies (electron microscopy observations), enzyme activities (Na+/K(+)-ATPase, Ca2+/Mg(2+)-ATPase, acetylcholinesterase and glucose-6-phosphatase), biochemical composition (lipid and protein analysis) and the tetrodotoxin sensitivity of the veratridine-induced gamma-aminobutyric acid (GABA) release. Isolating the SPM vesicles by the proposed procedure permits manipulating the ionic gradients across the membrane by changing the ion concentrations on either side or by utilizing specific ionophores. The vesicles retain their various activities, including their capacity for neurotransmitter uptake and release assays for at least 3 months, when preserved at -70 degrees C. Furthermore, the vesicles permit depicting the electrochemical gradients across the membranes into chemical and electrical components. We describe the use of the tetraphenylphosphonium cation (TPP+) to dissipate the membrane potential (delta psi) of the vesicles, while preserving ionic gradients. The characteristics of the lipid-soluble cation TPP+ allows a massive inflow of this cation into vesicular compartments and a consequent depolarization.

Topics: Animals; Brain; Cations; Electrolytes; Electrophysiology; gamma-Aminobutyric Acid; Indicators and Reagents; Male; Membrane Potentials; Neurosciences; Neurotransmitter Agents; Onium Compounds; Organophosphorus Compounds; Sheep; Synaptic Vesicles; Tetrodotoxin; Valinomycin

Isolated rat liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP+)-selective electrode. By flow cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP+. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-1 provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.

Topics: Adenosine Diphosphate; Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Female; Flow Cytometry; Fluorescent Dyes; Indicators and Reagents; Ion-Selective Electrodes; Ionophores; Membrane Potentials; Mitochondria, Liver; Onium Compounds; Organophosphorus Compounds; Phosphorylation; Potassium Chloride; Rats; Rats, Wistar; Reproducibility of Results; Sensitivity and Specificity; Valinomycin

Heart mitochondria contain a nNa+/Ca2+ antiport that participates in the regulation of matrix [Ca2+]. Based largely on a single study (Brand, M. D. (1985) Biochem. J. 229, 161-166), there has been a consensus that this antiport promotes the electroneutral exchange of two Na+ for one Ca2+. However, a recent study in our laboratory (Baysal, K., Jung, D. W., Gunter, K. K., Gunter, T. P., and Brierley, G. P. (1994) Am. J. Physiol. 266, C800-C808) has shown that the Na(+)-dependent efflux of Ca2+ from heart mitochondria has more energy available to it than can be supplied by a passive 2Na+/Ca2+ exchange. We have therefore re-examined Brand's protocols using fluorescent probes to monitor matrix pH and free [Ca2+]. Respiring heart mitochondria, suspended in KCl and treated with ruthenium red to block Ca2+ influx, extrude Ca2+ and establish a large [Ca2+]out:[Ca2+]matrix gradient. The extrusion of Ca2+ under these conditions is Na(+)-dependent and diltiazem-sensitive and can be attributed to the nNa+/Ca2+ antiport. Addition of nigericin increases the membrane potential (delta psi) and decreases delta pH to 0.1 or less, but has virtually no effect on the magnitude of the [Ca2+] gradient. Under these conditions a gradient maintained by electroneutral 2Na+/Ca2+ antiport should be abolished because the mitochondrial Na+/H+ antiport keeps the [Na+] gradient equivalent to the [H+] gradient. The [Ca2+] gradient is abolished, however, when an uncoupler is added to dissipate delta psi or when the exogenous electroneutral antiport BrA23187 is added. In addition, [Ca2+] influx via the nNa+/Ca2+ antiport in nonrespiring mitochondria is enhanced when delta psi is abolished. These results are consistent with Ca2+ extrusion by an electrophoretic antiport that can respond to delta psi but not with an electroneutral antiport.

Topics: Animals; Antiporters; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cattle; Cyclosporine; Diltiazem; Fluorescent Dyes; Hydrogen-Ion Concentration; Membrane Potentials; Mitochondria, Heart; Nigericin; Onium Compounds; Organophosphorus Compounds; Oxygen; Sodium; Sodium-Calcium Exchanger; Thermodynamics; Valinomycin

Bacillus licheniformis NCIB 6346 showed active accumulation of glucose which was inhibited by agents which affect the transmembrane proton gradient. Phosphotransferase (PTS) activity, identified as phosphoenolpyruvate-dependent phosphorylation of glucose, was found in cell extracts but could not be demonstrated in cells permeabilized with toluene when assays were conducted at pH 6.6. The same was true for mannitol and fructose phosphotransferase activities. Cells grown on fructose accumulated glucose at a slower rate than glucose-grown cells, and extracts prepared from them did not contain glucose PTS activity. Examination of the effects of analogs on glucose uptake and phosphorylation showed that 2-deoxyglucose was not a PTS substrate, but did markedly inhibit glucose uptake, with stronger inhibition in cells grown on fructose. Glucose accumulation by whole cells grown on glucose became less sensitive to the uncoupler tetrachlorosalicylanilide (TCS) as the pH was raised from 6.6 to 8.0, while in fructose-grown cells TCS was equally effective across this pH range. PTS activity was exhibited by toluene-treated cells at pH 7.5 and above, although the system itself in extracts was not affected by pH in the range of 5.0 to 8.0. The results are consistent with the presence of two glucose transport systems, one a PTS and the other operating by an alternative mechanisms, and suggest that the PTS in B. licheniformis may be regulated in a pH-dependent manner.

Topics: Bacillus; Biological Transport, Active; Cell Membrane Permeability; Energy Metabolism; Glucose; Hydrogen-Ion Concentration; Monosaccharide Transport Proteins; Onium Compounds; Organophosphorus Compounds; Phosphorylation; Phosphotransferases; Proton-Translocating ATPases; Salicylanilides; Sugar Phosphates; Valinomycin

The effects of several metabolic inhibitors on the uptake of tri-n-butylmethylammonium (TBuMA) were studied in isolated rat liver mitochondria, isolated rat hepatocytes and isolated perfused rat livers, in order to characterize further the mechanisms for carrier-mediated uptake and cellular accumulation of organic cations in the liver. Treatment of isolated hepatocytes with valinomycin, carbonylcyanide-m-chlorophenyl-hydrazone (CCCP), dinitrophenol, oligomycin or antimycin resulted in a rapid decrease in cellular ATP within 3 min of addition. The initial uptake rate of TBuMA was generally largely affected by these treatments. However, fructose at 10 mM had no effect at all on the uptake rate of the cation whereas cellular ATP was decreased to an extent comparable to that after treatment with the metabolic inhibitors. Consequently it was hypothesized that the metabolic inhibitors affected the initial cellular uptake rate of organic cations due to either altered intracellular sequestration (e.g. mitochondria) or alternatively to direct effects on the plasma membrane rather than by decreasing cellular ATP. Isolated rat mitochondria were shown to take up organic cations very efficiently. Accumulation in this organelle is probably driven by the negative membrane potential as measured by the uptake of the lipophilic cation [3H]tetraphenylphosphonium. Treatment of the isolated mitochondria with various metabolic inhibitors decreased the membrane potential in parallel to the effects on the uptake of TBuMA. Since mitochondria constitute a considerable intracellular volume, they may contribute largely to the storage of the organic cation in the hepatocyte. In isolated perfused livers, preloaded with either TBuMA or tetraphenylphosphonium (TPP+), the addition of valinomycin or CCCP leads to a marked backflux of the cations from the liver into the perfusion medium. This suggests strongly that a large part of the intracellular storage capacity is lost after metabolic inhibitor treatment, probably as the consequence of dissipation of the mitochondrial membrane potential. Since the metabolic inhibitors in contrast to TBuMA uptake did not decrease the initial uptake rate of TPP+ into isolated hepatocytes, it was concluded that mitochondrial uptake (mitochondria are the major storage sites for TPP+) is not an essential determinant of the initial uptake rate in intact hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations; Liver; Male; Mitochondria, Liver; Onium Compounds; Organophosphorus Compounds; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Valinomycin

The usefulness of ATP-depleted rat hepatocytes in transport studies was examined. ATP-depleted hepatocytes were prepared by incubating cell suspensions with 30 microM rotenone. In ATP-depleted hepatocytes, plasma membrane permeability was increased and mitochondrial membrane potential decreased, while both intracellular volume and pH remained normal. Furthermore, in the presence of valinomycin, the initial uptake rates of 3H-tetraphenyl phosphonium (TPP+) with varied medium concentrations of potassium were predicted according to the Goldman-Hodgkin-Katz equation, which demonstrated that a potassium diffusion potential could be produced in this system. Using the thus-characterized ATP-depleted cells, the uptake mechanism of taurocholate was investigated. In the presence of an inwardly directed Na gradient, the taurocholate uptake was markedly stimulated and bile acid was transiently accumulated at a concentration 3-times higher than at equilibrium ('overshoot') in ATP-depleted cells. No overshoot was observed in viable cells, however, which suggests that in ATP-depleted cells the Na gradient, a driving force for taurocholate uptake, decreased with time. In both viable and ATP-depleted cells, the relationship between medium concentrations of Na and the Na-dependent initial uptake rate were sigmoidal, and the Hill coefficients were close to 2. The Na-dependent initial uptake rate of taurocholate was stimulated by a valinomycin-induced inside negative potassium-diffusion potential in ATP-depleted cells, and the movement of a 'one plus' (as a net) charge was revealed by fitting the data to the Goldman-Hodgkin-Katz equation. These results support the hypothesis that sodium-coupled hepatic uptake of taurocholate occuthrough an electrogenic process with the stoichiometry of 2 Na: 1 taurocholate, although this issue is controversial. In the presence of an outwardly directed sodium gradient, efflux of taurocholate from ATP-depleted cells was not stimulated. Consequently, the physiological transport vector of taurocholate from blood to cell is not only due to the direction of the sodium gradient (blood to cell) but also to membraneous orientation of transport carriers. In conclusion, kinetic analysis using ATP-depleted hepatocytes allowed the formulation of a new approach to clarify the as yet unresolved issues concerning transport stoichiometry and the mechanism for vectorial transport of taurocholate.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Biological Transport; Cell Membrane Permeability; Cell Separation; Cells, Cultured; Hydrogen-Ion Concentration; Liver; Male; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Rats; Rats, Inbred Strains; Sodium; Taurocholic Acid; Valinomycin

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.

Topics: Animals; Cations; Cell Membrane Permeability; Electrophysiology; Energy Metabolism; Escherichia coli; Fluorescent Dyes; Indoles; Mitochondria, Liver; Nigericin; Onium Compounds; Organophosphorus Compounds; Pyridinium Compounds; Rats; Tetraphenylborate; Trialkyltin Compounds; Valinomycin

The rate of protonophore-mediated decay of pH gradient across lipid vesicular membranes was found to be enhanced by orders of magnitude by valinomycin-K+. Experiments in the presence of gramicidin have shown that the observed rate enhancement by valinomycin-K+ is not due to collapse of the diffusion potential alone. The enhancement of the rate showed hyperbolic dependence on the concentration of valinomycin. Rate enhancement was observed in the presence of the membrane permeant cation tetraphenylphosphonium (TTP+) also. Several factors which might enhance the intrinsic H+ conductivity of protonophores were analyzed. The level of partitioning of the protonophore into the membrane and the pK of membrane-bound protonophores were measured. Valinomycin-K+ did not alter both these parameters significantly. TPP+ increased the partitioning of protonophores and decreased the pK values of membrane-bound protonophores. However, these changes were too small to explain the observed rate enhancements. We suggest that valinomycin-K+ and TPP+ enhance the H+ conductivity of protonophores by increasing the permeability of the ionized form of protonophores by forming an ion pair.

Topics: Boron Compounds; Cations; Cell Membrane Permeability; Gramicidin; Hydrogen; Hydrogen-Ion Concentration; Kinetics; Onium Compounds; Organophosphorus Compounds; Potassium; Tetraphenylborate; Valinomycin

An electrode sensitive to tetraphenyl phosphonium (TPP+) was applied to monitor the changes in the membrane potential of the ciliate Blepharisma japonicum during the process of cell elongation. Uptake of TPP+ by Blepharisma cells was observed when they were incubated in a medium containing 2 x 10(-6)M TPP+. Subsequent light stimulation (3,000 lux) led to an efflux of accumulated TPP+ from the cells, indicating that depolarization of the plasma membrane occurred with the concomitant cell elongation. TPP+ efflux was always observed whenever cell elongation was induced in response to various stimuli: valinomycin (10(-6)M), K+ (30 mM), Co2+ (5 mM), and Ca2+ (5 mM). Furthermore, elongation was also evoked by electrical stimulation. These observations indicate that membrane depolarization is involved in the process of cell elongation in Blepharisma.

Topics: Animals; Calcium; Cell Membrane; Cell Movement; Cells, Cultured; Ciliophora; Cobalt; Electric Stimulation; Electrodes; Light; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Valinomycin

The net influx of L-arginine (JARG) was employed as an indicator of the membrane potential in human fibroblasts. Cell depolarization, obtained by increasing [K+]out, decreased both JARG and the net influx of the lipid soluble cation tetraphenylphosphonium (JTPP), a probe of membrane potential. JTPP, but not JARG, was influenced by the mitochondrial potential and exhibited a component dependent on intracellular and/or extracellular binding. JARG was sensitive to changes in the membrane potential induced by Na+-dependent transport of L-proline or by the activity of Na+-K+-ATPase. In the presence of 50 microM valinomycin, JARG was markedly influenced by the distribution ratio of K+ in a range of [K+]out from 1.5 to 100 mM. In this range of [K+]out, membrane potential (Em) varied from -90 to -23 mV, and calibration of JARG vs. the membrane potential yielded a linear relationship. These results indicate the following: 1) that the net influx of TPP+ is not a reliable indicator of membrane potential in cultured human fibroblasts; 2) that in the same cells the net influx of L-arginine can be employed as an index of membrane potential; 3) that in a range of Em from -23 to -90 mV the activity of system y+ (the membrane agency devoted to L-arginine transport in cultured human fibroblasts) exhibits no saturation of potential-dependent activation of transport.

Topics: Arginine; Biological Transport; Cells, Cultured; Fibroblasts; Humans; Kinetics; Membrane Potentials; Mitochondria; Onium Compounds; Organophosphorus Compounds; Potassium; Proline; Sodium; Sodium-Potassium-Exchanging ATPase; Valinomycin

The role of cellular energy, and mainly that of electrical transmembrane potential, in cadmium fixation by a marine pseudomonad suspended in a mineral medium was investigated by studying the effects of ionophores. Although fixation of cadmium by cells was generally less when respiratory activity was inhibited, it was not affected by a reduction of the transmembrane electrical potential delta psi in mureinoplasts. These observations strongly suggest that cadmium fixation in this isolate was not the result of a delta psi-dependent active transport.

Topics: Antineoplastic Agents; Biological Transport, Active; Cadmium; In Vitro Techniques; Ionophores; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Pseudomonadaceae; Valinomycin

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.

Topics: Algorithms; Chlorides; Cholesterol; Lipid Bilayers; Liposomes; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Rubidium; Sodium; Trityl Compounds; Valinomycin

Ferrous iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out in a well-defined salt solution termed "modified Hanks solution" at both high iron concentrations (LAFIUS conditions) and low concentrations (HAFIUS conditions). Various divalent metals, Mn2+, Zn2+, Ni2+ and Cu2+, inhibited iron uptake under HAFIUS conditions in a non-competitive manner, and in a pseudo-competitive manner under LAFIUS conditions. Cr2+ had no effect. Co2+ inhibited iron uptake competitively under HAFIUS conditions. Metabolic affectors that inhibited iron uptake both under HAFIUS and LAFIUS conditions were: tetraphenylphosphonium chloride, diethylstilbesterol, vanadate, carbonylcyanide-m-chlorophenyl-hydrazone, and a mixture of valinomycin and nigericin. Substances that stimulated iron uptake were KCl, valinomycin, and nigericin. Iron uptake under LAFIUS conditions in piperazine-buffered modified Hanks solution was higher than that in the acetate-buffered solution, and acetate inhibited iron uptake in the piperazine buffer. HAFIUS showed no difference. It is concluded that iron uptake in bifidobacteria is driven by an ATPase-dependent proton-motive force and that both the pH gradient and membrane potential are involved in this process. Mn2+, Zn2+, Ni2+, and Cu2+ may be transported via LAFIUS, but not HAFIUS. HAFIUS may transport only Co2+ in addition to Fe2+.

Topics: Bifidobacterium; Biological Transport; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations, Divalent; Copper; Diethylstilbestrol; Ferrous Compounds; Manganese; Nickel; Nigericin; Onium Compounds; Organophosphorus Compounds; Potassium Chloride; Protons; Solutions; Valinomycin; Vanadates; Vanadium; Zinc

The cell-to-medium distribution ratios at steady state of L-arginine (RArg) and of the lipid-soluble cation tetraphenylphosphonium (RTPP) were studied as a function of the membrane potential (Em) in adult human fibroblasts. The relationship between RArg and Em was qualitatively similar to that of RTPP and Em. Quantitatively, RArg and RTPP differed in that 1) RTPP was much greater than RArg when Em was near zero, indicating a significant binding component in the uptake of TPP+ but not of L-arginine, and 2) after a correction for binding when Em is near zero, RTPP was still greater than RArg so that RT/F . ln RTPP exceeded RT/F . ln RArg by 10-25 mV. The pattern of the redistribution of accumulated TPP+ and arginine after an alteration of Em was identical. In null-point experiments, the external [K+] for which there were no changes in cellular TPP+ or L-arginine in the presence of high valinomycin (the null points) were very similar for the two probes. Em calculated from the null-point measurements (-70(-)-80 mV) was also very similar to RT/F . ln RArg and thus smaller than RT/F.ln RTPP. It was concluded that 1) there was an additional TPP+ binding as cellular [TPP] rose in response to more negative membrane potentials, 2) the transport system for L-arginine in these cells (system y+) operates as a facilitated diffusion system driven by the membrane potential, and 3) in some circumstances, L-arginine could be employed as a probe of Em.

Topics: Arginine; Calibration; Cells, Cultured; Fibroblasts; Homeostasis; Humans; Membrane Potentials; Methods; Onium Compounds; Organophosphorus Compounds; Skin; Time Factors; Valinomycin

C6 glioblastoma cells in culture were employed to isolate plasma membrane vesicles. After disruption of the glioblastoma cells by homogenization, membrane fractions were obtained by centrifugation on a discontinuous Ficoll density gradient. Fragmented membranes were found mainly in vesicular form. Transport of glycine has been demonstrated in membrane vesicles, using artificially imposed ion gradients as the sole energy source. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a Cl- gradient (out greater than in) when the other essential ion is present. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of ionophore valinomycin and anions with different permeabilities. The kinetic analysis shows that glycine is accumulated by two systems with different affinities.

Topics: Biological Transport; Cell Line; Cell Membrane; Glioma; Glycine; Kinetics; Nigericin; Onium Compounds; Organophosphorus Compounds; Sodium-Potassium-Exchanging ATPase; Valinomycin

The plasmamembrane potential (delta psi p) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, AR exp) was measured by adding 2 microM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to AR exp, evaluated by adding carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or 2,4-dinitrophenol (DNP), was apparently negligible in standard cultures, AR exp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong AR exp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit AR exp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to AR exp values, delta psi p of these cells was then measured; this potential varying from -65 mV to -16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of delta psi p on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (PK).

Topics: Animals; Cell Count; Cell Line; Cell Membrane Permeability; Culture Media; Leukemia, Erythroblastic, Acute; Mathematics; Membrane Potentials; Mice; Mitochondria; Onium Compounds; Organophosphorus Compounds; Potassium; Valinomycin

When Schistosoma mansoni cercariae penetrate the skin of the mammalian host they rapidly pass from fresh water to a high salt physiologic environment and transform into schistosomula. Following this transition, the parasites migrate from the skin to the lungs during which time they change from being highly susceptible to immune attack to being refractory, as measured by in vitro cytotoxicity assays. In this study, in vivo or in vitro schistosomula of different ages were examined for developmentally linked changes in membrane function which might correlate with the attainment of the resistant state. In particular, alterations in the distribution of tetraphenylphosphonium (TPP+), a synthetic lipophilic cation which shows a potential dependent partition across membranes, were followed. Three-hour-old schistosomula, which are greater than 75% susceptible to antibody-dependent complement-mediated attack or lymphokine-activated macrophage-mediated cytotoxicity, acquired TPP+ at a similar rate and steady state level to 5-day-old lung worms, which were completely resistant to both these effector mechanisms. The addition of ouabain, a Na+/K+-ATPase inhibitor, caused a 50% decrease in both the rate and steady state of TPP+ uptake by 3 h parasites but had little effect on these parameters in lung worms. Valinomycin, a K+-ionophore, completely inhibited TPP+ influx in both stages. The characteristics of TPP+ efflux from 3-h and 5-day-old parasites preloaded with the cation were found to be dissimilar. Whereas 30% of acquired TPP+ was lost from lung worms within 2 h, only 10% of acquired cation was released from 3-h schistosomula during the same period.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adaptation, Physiological; Animals; Biological Transport, Active; Cytotoxicity, Immunologic; Kinetics; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Ouabain; Schistosoma mansoni; Valinomycin

The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K+. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), X-AG (for anionic amino acids), and y+ (for cationic amino acids). High doses (100 microM) of the K+-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y+. Transport systems L (for neutral amino acids) and x-C (for anionic amino acids) are insensitive to changes in extracellular K+ or to valinomycin. System X-AG is inhibited by the addition of 100 microM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x-C is potential-independent and (b) the activity of systems A, ASC, X-AG and y+ is sensitive to alterations of external [K+] associated to changes in membrane potential.

Topics: Amino Acids; Aminoisobutyric Acids; Aspartic Acid; Biological Transport, Active; Body Water; Female; Fibroblasts; Humans; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Pregnancy; Valinomycin

Accumulation of gentamicin by gentamicin-susceptible Staphylococcus aureus was examined by using the ionophorous antibiotic, valinomycin and the protontranslocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The effects of these inhibitors on the transmembrane electrical potential (delta psi) were determined by measuring the equilibrium distribution of the tetraphenylphosphonium ion. The results indicate a direct correlation between delta psi and the extent of gentamicin uptake. However, under conditions where a significant delta psi existed across the plasma membrane, uptake of gentamicin was negligible. A threshold delta psi may thus be required to initiate gentamicin uptake. The proposed threshold delta psi appears to vary depending upon the external concentration of gentamicin.

Topics: Dicyclohexylcarbodiimide; Gentamicins; Membrane Potentials; Nigericin; Onium Compounds; Organophosphorus Compounds; Potassium Chloride; Staphylococcus aureus; Time Factors; Valinomycin

Topics: Biological Transport, Active; Carbohydrate Metabolism; Cell Membrane; Cell Membrane Permeability; Electrochemistry; Indicators and Reagents; Kinetics; Membrane Potentials; Methylglucosides; Onium Compounds; Organophosphorus Compounds; Potassium; Sodium; Valinomycin

The relationship between alterations in transmembrane potential, cell volume, and phospholipid fatty acid turnover has been examined in human erythrocytes by treating the cells with the monovalent cation ionophore valinomycin. Valinomycin increases the cellular uptake of tetra[3H]phenylphosphonium ion by erythrocytes, indicating membrane hyperpolarization, and causes net loss of potassium chloride and water from the cells leading to a decrease in cell volume. Treatment of erythrocytes with valinomycin also enhances incorporation of [9, 10-(3)H]oleic acid into phospholipids, primarily diacylphosphatidylethanolamine. After replacing intracellular chloride with sulfate and treating cells with the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate, exposure to valinomycin results in uptake of tetra[3H]phenylphosphonium ion and stimulation of [9, 10-(3)H]oleic acid incorporation, but, because anion efflux is prevented, no decrease in cell volume occurs. When tetra[3H]phenylphosphonium ion uptake is also prevented by suspending these cells in 125 mM KCl to dissipate the transmembrane potassium gradient, valinomycin still enhances [9, 10-(3)H] oleic acid incorporation into phospholipid. These results suggest that the presence of valinomycin in the membrane directly alters phospholipid fatty acid turnover and that some of the effects of this ionophore on cellular function previously attributed to alterations in transmembrane potential or cellular potassium content may instead be due to altered phospholipid turnover. Since it is possible that valinomycin may directly perturb phospholipid fatty acid turnover in other cells, the possibility that valinomycin-induced alterations in cellular function are due to altered phospholipid turnover rather than membrane hyperpolarization or altered potassium content should be considered in the interpretation of studies employing this ionophore.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Erythrocyte Indices; Erythrocytes; Fatty Acids; Humans; Membrane Lipids; Membrane Potentials; Oleic Acid; Oleic Acids; Onium Compounds; Organophosphorus Compounds; Phosphatidylethanolamines; Phospholipids; Valinomycin

Starved whole cells of the obligately alkalophilic Bacillus firmus RAB synthesize ATP upon addition of L-malate at pH 9.0 as expected of an aerobic organism that grows oxidatively on nonfermentable carbon sources at pH values as high as 11.0. The current study was a detailed examination of the perplexing inability of such cells to exhibit ATP synthesis in response to a valinomycin-mediated potassium diffusion potential at pH 9.0. While there were minor differences in the patterns of generation of the potential and the proton influx that accompanies its generation in the three different buffering systems employed, the magnitude of the transmembrane electro-chemical potential of protons was at least as high as pH 9.0 as at pH 7.0. Nevertheless, a diffusion potential consistently energized ATP synthesis at pH 7.0 but not at 9.0; these findings were independent of the presence or absence of Tris or of Na+. By contrast, the artificial electron donor ascorbate, in the presence of phenazine methosulfate, energized ATP synthesis by the starved whole cells at both pH values. The same phenomenon, i.e., efficacy of a respiration-derived potential but not of a diffusion potential at pH 9.0, was demonstrated in ADP + Pi-loaded membrane vesicles. On the other hand, electrogenic Na+-coupled solute transport could be energized by both ascorbate/phenazine and methosulfate and a diffusion potential in the vesicles at pH 9.0. The results are discussed in connection with models of a localized path of proton flow between proton pumps and the ATP synthase.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aminoisobutyric Acids; Bacillus; Diffusion; Hydrogen-Ion Concentration; Onium Compounds; Organophosphorus Compounds; Phosphates; Potassium; Valinomycin

Quantitative studies of MCF-7 cells (derived from human breast adenocarcinoma) and CV-1 cells (from normal African green monkey kidney epithelium), using the permeant cationic compound tetraphenylphosphonium (TPP), in conjunction with fluorescence microscopy using rhodamine 123 (Rh123), indicate that the mitochondrial and plasma membrane potentials affect both uptake and retention of these compounds. Under conditions that depolarize the plasma membrane, uptake and retention of TPP and Rh123, driven only by the mitochondrial membrane potential, is greater in MCF-7 than in CV-1. An ionophore that dissipates the mitochondrial membrane potential of MCF-7 cells causes them to resemble CV-1 cells by decreasing uptake and retention. Hyperpolarizing the mitochondrial membrane of CV-1 increases accumulation and prolongs retention; hyperpolarization of the plasma membrane further heightens this effect, causing the uptake of CV-1 cells to resemble that of MCF-7 cells even more closely. The greater uptake and retention by MCF-7 appears to be a consequence of elevated mitochondrial and plasma membrane potentials. The plasma membrane potential affects mitochondrial retention of TPP and Rh123 and its role in enhancing the effect of a difference in mitochondrial membrane potential is explained.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Line; Chlorocebus aethiops; Female; Humans; Kidney; Membrane Potentials; Mitochondria; Nigericin; Onium Compounds; Organophosphorus Compounds; Ouabain; Potassium; Rhodamine 123; Rhodamines; Sodium; Valinomycin; Xanthenes

Methods are described which demonstrate the use of unidirectional influx of 14C-tetraphenylphosphonium (14C-TPP+) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential (delta psi) and Na+-dependent sugar influx was determined for these cells at various Na+ and alpha-methylglucoside (alpha-MG) concentrations. The results show a high degree of delta psi dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of delta psi. No transinhibition by intracellular sugar (40 mM) can be detected. Sugar influx in the absence of Na+ is insensitive to 1.3 mM phlorizin and independent of delta psi. The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na+ binding is the potential-dependent step (Na+ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.

Topics: Animals; Biological Transport, Active; Cell Membrane; Chickens; Epithelium; Intestinal Mucosa; Intestine, Small; Intracellular Fluid; Kinetics; Membrane Potentials; Methylglucosides; Methylglycosides; Onium Compounds; Organophosphorus Compounds; Phlorhizin; Potassium; Sodium; Valinomycin

Sea urchin sperm motility is activated by a Na+-dependent increase of internal pH. A flagellar preparation was used in the present study to investigate this ionic mechanism. Using 22Na and a pH electrode, the stoichiometry of Na+ uptake to H+ release in the isolated flagella was found to be 1.09 +/- 0.11. Reversing the Na+ gradient induced reacidification of the intraflagellar pH as measured by [14C]methylamine, while reversal of the H+ gradient resulted in a Na+ efflux. Furthermore, a parallel inhibition of both ionic movements was observed with increasing external [K+]. These results indicate that Na+ and H+ are coupled through an exchanger. Measurements of the membrane potential (psi) with [3H]tetraphenylphosphonium showed depolarization by K+, suggesting its inhibitory effect on the exchanger is through changes in psi. This is further supported by the following experiments. (a) Cs+ by itself had little effect on either psi or the Na+/H+ exchange, but in the presence of the ionophore valinomycin it depolarized psi and inhibited the exchange. (b) Tetraphenylphosphonium a highly permeant cation, at 2.5 mM caused depolarization and inhibition of the exchange, and these effects were reversible by repolarization of psi with valinomycin. The inhibitory effect of depolarization was not due to the electrogenicity of the exchange since both directions of the exchange were inhibited. It is proposed that the flagellar exchange is basically a electroneutral process but has a charged regulatory component (a gate or a conformational change) which confers the observed potential sensitivity.

Topics: Animals; Carrier Proteins; Flagella; Hydrogen-Ion Concentration; Kinetics; Male; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Sea Urchins; Sodium; Sodium-Hydrogen Exchangers; Sperm Motility; Spermatozoa; Valinomycin

This study explored the role of the proton motive force in the processes of DNA binding and DNA transport of genetic transformation of Bacillus subtilis 168 strain 8G-5 (trpC2). Transformation was severely inhibited by the ionophores valinomycin, nigericin, and 3,5-di-tert-4-hydroxybenzylidenemalononitrite (SF-6847) and by tetraphenylphosphonium. The ionophores valinomycin and nigericin also severely inhibited binding of transforming DNA to the cell envelope, whereas SF-6847 and carbonylcyanide-p-trifluoromethoxyphenylhydrazone hardly affected binding. The proton motive force, therefore, does not contribute to the process of DNA binding, and valinomycin and nigericin interact directly with the DNA binding sites at the cell envelope. The effects of ionophores, weak acids, and tetraphenylphosphonium on the components of the proton motive force and on the entry of transforming DNA after binding to the cell envelope was investigated. DNA entry, as measured by the amount of DNase I-resistant cell-associated [3H]DNA and by the formation of DNA breakdown products, was severely inhibited under conditions of a small proton motive force and also under conditions of a small delta pH and a high electrical potential. These results suggest that the proton motive force and especially the delta pH component functions as a driving force for DNA uptake in transformation.

Topics: Bacillus subtilis; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; DNA, Bacterial; Hydrogen-Ion Concentration; Membrane Potentials; Nigericin; Nitriles; Onium Compounds; Organophosphorus Compounds; Protons; Transformation, Bacterial; Valinomycin

The membrane potential across the cytoplasmic membrane of EDTA-treated cells of a K+ transport-negative mutant of Escherichia coli K-12 was estimated from the equilibrium distribution of different lipid-soluble cations. With glucose as a substrate and at low K+ out, the membrane potential calculated from the distribution ratio of 86Rb+ in the presence of valinomycin (delta psi rb+) was considerably higher than that indicated by the [3H]tetraphenylphosphonium cation (delta psi TPP+). The lipid-soluble anion phenyldicarbaundecaborane (PCB-) increased delta psi TPP+ close to delta psi Rb+. To investigate whether these results were due to different binding of the cations to cellular components, residual Rb+ and TPP+ uptake was measured in cells permeabilized with 5% n-butanol (by volume). In those cells the distribution ratios or Rb+, K+ and Na+ approached a value of 4, indicating that the uptake of all three ions was driven by a residual negative surface potential or transmembrane Donnan potential (internally negative). The distribution ratio of TPP+ was 3--4-times higher than that of other cations and up to 10 mM TPP+ out was almost independent of the added TPP+ concentration. This extra uptake presumably represents binding of TPP+ to the cellular membranes. Thus, at pH 7.5, delta psi Rb+ was about 180--200 mV, whereas after correction for binding delta psi TPP+ was 110--150 and 150--170 mV in the absence and presence of PCB-, respectively. It is proposed that TPP+ indicates too low a potential, because by its strong binding it decreases the negative surface potential of the cytoplasmic membrane, and thereby inhibits its own further uptake. This is taken to mean that TPP+ distribution can be used as a qualitative probe only for the bacterial membrane potential.

Topics: Cell Membrane; Edetic Acid; Escherichia coli; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Membrane Potentials; Mutation; Onium Compounds; Organophosphorus Compounds; Potassium; Rubidium; Valinomycin

The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H+ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H+ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10 mM glucose an outward-directed H+ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H+ reverses the direction of the H+ concentration gradient. Changes in absorbance of the voltage-sensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H+ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.

Topics: Acridine Orange; Animals; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane; Coloring Agents; Glucose; Glycolysis; Hydrogen-Ion Concentration; Kinetics; Mice; Onium Compounds; Organophosphorus Compounds; Protons; Salicylanilides; Valinomycin

The magnitude of delta psi (membrane potential), delta pH (pH gradient), lactose accumulation and cytoplasmic volume have been determined over a range of experimental conditions. A study of two probes of delta pH, benzoate and dimethyloxazolidene-2,4-dione (DMO), and four probes of delta psi, Rb+, K+, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiodicarbocyanine iodide, has been carried out. Benzoate and DMO are shown to be equivalent at pH values above the pK of DMO, but the latter may be less accurate below this pH. The cations TPP+ and Rb+ were found, by a number of criteria, to be equivalent, and TPP+ may be used in cells not pretreated with EDTA. These studies are an essential prerequisite to the use of TPP+ as a quantitative probe in untreated cells.

Topics: Escherichia coli; Hydrogen-Ion Concentration; Indicators and Reagents; Lactose; Membrane Potentials; Methods; Onium Compounds; Organophosphorus Compounds; Protons; Rubidium; Thermodynamics; Valinomycin

Using the penetrating ions of tetraphenylphosphonium (TPP+) and tetraphenylborone (TPB-), the membrane potential of the Bacillus subtilis and Escherichia coli cells was shown that the TPP+ absorption by the cells is an energy-coupled process. The TPB- anions are released from the cells after addition of an energy substrate. The value of the membrane potential calculated from the distribution pattern of the penetrating ions in the cells and the incubation medium lies within the interval of --100--150 mV (intracellular negative electric potential). The value of the membrane potential strongly depends on pH of the incubation medium; our attempts to measure the membrane potential in the E. coli cells at ph 6.0 were unsuccessful; however, at pH 8.5 it was found to be equal to --100 mV. Treatment of the cells with nigericin partially prevents the decrease of the membrane potential in an acidic medium and increases the potential in neutral and alkaline media. The formation of the membrane potential is suppressed by valinomycin and gramicidine, as well as by the oxidative phosphorylation uncouplers; the inhibiting effect of valinomycin requires the presence of K+ in the incubation medium. The membrane potential of the B. subtilis cells is insensitive to the effect of cyanide in the absence of arsenate. It is concluded that the membrane potential of B. subtilis and E. coli is formed both via respiration and by hydrolysis of intracellular ATP.

Topics: Bacillus subtilis; Boron Compounds; Escherichia coli; Gramicidin; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Tetraphenylborate; Valinomycin

Recently an energy-recycling model was proposed that postulates the generation of an electrochemical gradient in fermentative bacteria by carrier-mediated excretion of metabolic end products in symport with protons. In this paper experimental support for this model is given. In batch cultures of Streptococcus cremoris with glucose as the sole energy source the maximal specific growth rate decreased by 30% when the external lactate concentration was decreased from 50 to 90 mM. In the same range of external lactate concentrations the molar growth yield Y for glucose as measured in energy-limited chemostat cultures also showed a 30% drop. From Y max lactose values of S. cremoris grown in the presence and absence of added lactate it was calculated that the net energy gain from the lactate efflux system was at least 12%. Lactate efflux from de-energized cells loaded with lactate could drive the uptake of leucine. This uptake was sensitive to carbonylcyanide p-trifluoromethoxyphenylhydrazone and was only partly inhibited by dicyclohexylcarbodiimide (DCCD). The limited inhibition by DCCD of lactate-induced leucine uptake indicates that ATP hydrolysis was not the driving force for transport of leucine. Uptake studies with the lipophilic cation tetraphenylphosphonium demonstrated that lactate efflux increased the electrical potential across the membrane by 51 mV. The generation of an electrical potential by lactate efflux and the demonstration of a potassium efflux-induced uptake of lactate indicates that lactate is translocated across the membrane by a symport system with more than one proton.

Topics: Biological Transport, Active; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Dicyclohexylcarbodiimide; Electrophysiology; Energy Metabolism; Lactates; Leucine; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Protons; Streptococcus; Valinomycin

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.

Topics: Benzoates; Cell Membrane; Dialysis; Hydrogen-Ion Concentration; Membrane Potentials; Methylamines; Methylphenazonium Methosulfate; Micrococcus; Onium Compounds; Organophosphorus Compounds; Valinomycin

Sarcolemmal membrane vesicles isolated from canine ventricular tissue accumulate calcium through the sodium-calcium exchange system when an outwardly directed sodium gradient is generated across the vesicle membrane. Moreover, calcium uptake under these conditions is accompanied by the transient accumulation of the lipophilic cation tetraphenylphosphonium. Since the distribution of tetraphenylphosphonium across biological membranes reflects the magnitude and direction of transmembrane potential differences and the characteristics of the transient accumulation of this cation closely resemble those of sodium-calcium exchange activity, it is concluded that a membrane potential, interior negative, is produced during calcium accumulation through the exchange system. Thus, the operation of the sodium-calcium exchange system generates a current in cardiac membrane vesicles, suggesting that three or more sodium ions exchange for each calcium ion.

Topics: Biological Transport, Active; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Heart; Kinetics; Membrane Potentials; Myocardium; Onium Compounds; Organophosphorus Compounds; Sodium; Valinomycin