monensin and tetraphenylphosphonium

Tetraphenylphosphonium (TPP+) is a permeant lipophilic cation that accumulates in cultured cells and tissues as a function of the electrical membrane potential across the plasma membrane. This study was undertaken to determine whether TPP+ can be used for assessing membrane potential in intact lenses in organ culture.. Rat lenses were cultured in media containing 10 microM TPP+ and a tracer level of 3H-TPP+ for various times. 3H-TPP+ levels in whole lenses or dissected portions of lenses were determined by liquid scintillation counting. Ionophores, transport inhibitors, and neurotransmitters were also added to investigate their effects on TPP+ uptake. RESULTS. Incubation of lenses in low-K+ balanced salt solution and TC-199 medium, containing physiological concentrations of Na+ and K+, led to a biphasic accumulation of TPP+ in the lens that approached equilibrium by 12 to 16 hours of culture. The TPP+ equilibrated within 1 hour in the epithelium but penetrated more slowly into the fiber mass. The steady state level of TPP+ accumulation in the lens was depressed by 90% when the lenses were cultured in a medium containing high K+. The calculated membrane potential for the normal rat lens in TC-199 was -75 +/- 3 mV. Monensin (1 microM) and nigericin (1 microM), Na+H+ and K+H+ exchangers respectively, as well as the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 microM) and the calcium ionophore A23187 (10 microM), abolished TPP+ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 microM decreased TPP+ accumulation in the lens, but this effect could be prevented by simultaneous application of 1 mM atropine.. TPP+ accumulation can be used as an indicator of changes in membrane potential in intact lenses, but because of the long time required to reach steady state, its utility is limited. The slow accumulation of TPP+ and its slow efflux from the lens under conditions known to depolarize membranes are consistent with a diffusion barrier in the deep cortex and nucleus of the lens.

Topics: Animals; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Culture Media; Female; Indicators and Reagents; Lens, Crystalline; Male; Membrane Potentials; Monensin; Monitoring, Physiologic; Nigericin; Onium Compounds; Organ Culture Techniques; Organophosphorus Compounds; Rats; Rats, Sprague-Dawley

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.

Topics: Animals; Calcium; Cell Membrane; Glyceraldehyde; Insulinoma; Ion Pumps; Male; Monensin; Onium Compounds; Organophosphorus Compounds; Rats; Rats, Inbred Strains; Sodium; Sodium-Potassium-Exchanging ATPase

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+.

Topics: Arylsulfonates; Biological Transport; Buffers; Carrier Proteins; Hydrogen; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Monensin; Onium Compounds; Organophosphorus Compounds; Sodium; Sodium-Hydrogen Exchangers

The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells. The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only. The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids. Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin. In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of [3H]TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions. On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of [3H]TPP+-accumulation. These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential.

Topics: Amino Acids; Cell Line; Culture Media; Humans; Leukemia, Erythroblastic, Acute; Membrane Potentials; Monensin; Onium Compounds; Organophosphorus Compounds; Ouabain; Rubidium Radioisotopes; Tumor Cells, Cultured

Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanide m-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Non-electrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (delta psi). The data could be explained by a delta psi threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of delta psi preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of delta psi induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP acts via specific sites on the surface of transformed cells.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane Permeability; Cell Transformation, Neoplastic; Cells, Cultured; Ionophores; Kinetics; Mice; Monensin; Nigericin; Onium Compounds; Organophosphorus Compounds; Potassium; Sodium; Uridine

Serum stimulates embryonic avian skeletal muscle growth in vitro and the growth-related processes of amino acid transport and protein synthesis. Serum also stimulates myotube Na pump activity (measured as ouabain-sensitive rubidium-86 uptake) for at least 2 h after serum addition. Serum-stimulated growth depends on this Na pump activity since ouabain added at the same time as serum totally inhibits the growth responses. The relationship of myotube growth, Na pump activity, and transmembrane potential was studied to determine whether serum-stimulated Na pump activation and growth are coupled by long-term membrane hyperpolarization. When myotube amino acid transport and protein synthesis are prestimulated by serum, ouabain was found to have little inhibitory effect, indicating that the already stimulated growth-related processes are not tightly coupled to continued Na pump activity. Serum-stimulated protein synthesis is tightly coupled to Na pump activity, but only during the first 5-10 min after serum addition. When myotube transmembrane potentials were measured using the lipophilic cation tetraphenylphosphonium, serum at concentrations that stimulate myotube growth and Na pump activity was found to have little effect on the cell's transmembrane potential. Furthermore, partial depolarization of the myotubes with 12- to 55-mM extracellular potassium does not prevent serum stimulation of myotube growth. Monensin was found to hyperpolarize the myotubes, but causes myotube atrophy. These results indicate that although Na pump activity is associated with initiation of serum-stimulated myotube growth, continued Na pump activity is not essential, and there is little relationship between myotube growth and the myotube's transmembrane potential.

Topics: Amino Acids; Animals; Cells, Cultured; Chick Embryo; Membrane Potentials; Monensin; Muscle Development; Muscle Proteins; Onium Compounds; Organophosphorus Compounds; Ouabain; Potassium; Sodium; Sodium-Potassium-Exchanging ATPase

Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct intracellular recording techniques and by distribution studies with the lipophilic cation [3H]-tetraphenylphosphonium+ (TPP+) [Lichtshtein, D., Kaback, H.R. & Blume, A.J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654]. The effect is not observed with cells suspended in high K+ medium, is dependent upon the presence of Na+ externally, and the concentration of monensin that induces half-maximal stimulation of TPP+ accumulation is approximately 1 microM. The ionophore also causes rapid influx of Na+, a transient increase in intracellular pH, and a decrease in extracellular pH, all of which are consistent with the known ability of monensin to catalyze the transmembrane exchange of H+ for Na+. Although ouabain has no immediate effect on the membrane potential, the cardiac glycoside completely blocks the increase in TPP+ accumulation observed in the presence of monensin. Thus, the hyperpolarizing effect of monensin is mediated apparently by an increase in intracellular Na+ that acts to stimulate the electrogenic activity of the Na+,K+-ATPase. Because monensin stimulates TPP+ accumulation in a number of other cultured cell lines in addition to NG108-15, the techniques described may be of general use for studying the Na+,K+ pump and its regulation in situ.

Topics: Animals; Biological Transport; Cell Line; Furans; Glioma; Hybrid Cells; Kinetics; Membrane Potentials; Mice; Monensin; Neuroblastoma; Onium Compounds; Organophosphorus Compounds; Ouabain; Potassium; Rats; Sodium