carbocyanines and tetraphenylphosphonium

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

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.

Topics: Benzothiazoles; Carbocyanines; Coloring Agents; Deuterium; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Membrane Potentials; Molecular Conformation; Onium Compounds; Organophosphorus Compounds; Phosphatidylcholines; Phosphatidylglycerols; Phosphorylcholine; Tetraphenylborate

The effect of elevated temperature on transmembrane potential was studied in Chinese hamster ovary cells in vitro using tetraphenylphosphonium cation (TPP+) and 3,3'-dipentyloxacarbocyanine [Di-O-C5(3)], two unrelated lipophilic cation probes that equilibrate across the plasma membrane according to the transmembrane potential. Uptake of TPP+ was measured using a tritium-labeled probe and the uptake of the fluorescent probe Di-O-C5(3) was measured by flow cytometry. The Nernst equation was used to calculate transmembrane potential. The absolute values obtained for transmembrane potential at 37 degrees C using the two probes were different, but qualitatively similar results were obtained using either probe in the hyperthermia studies. Transmembrane potential measured at 43 and 45 degrees C was at least 20% higher than that measured at 37 degrees C, and the difference was statistically significant (P = 0.025 and P less than 0.01, respectively). The hyperpolarization induced by exposure to 45 degrees C persisted temporarily after cells had been returned to 37 degrees C. The hyperpolarization at 37 degrees C associated with a previous exposure to hyperthermia was maximal after cells had been held at 45 degrees C for 2.0 min, and fell to normal levels after 15.0 min at 37 degrees C.

Topics: Animals; Carbocyanines; Hot Temperature; Indicators and Reagents; Membrane Potentials; Onium Compounds; Organophosphorus Compounds

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.

Topics: Acetylcholine; Adrenal Medulla; Animals; Carbocyanines; Catecholamines; Cattle; Cell Membrane; Chromaffin System; Decamethonium Compounds; Fluorescent Dyes; Ionophores; Kinetics; Membrane Potentials; Nicotine; Onium Compounds; Organophosphorus Compounds; Potassium; Quinolines; Receptors, Cholinergic; Veratridine

Effects of three probes for measuring membrane potential, tetraphenylphosphonium (TPP+), rhodamine 6G and 3,3'-dipropylthiocarbocyanine (diS-C3-(5)) on energy metabolism in synaptosomes were investigated. None of the three probes had any effect on lactate production in synaptosomes. TPP+ and rhodamine 6G did not inhibit the respiration of synaptosomes with pyruvate and succinate as exogenous substrate and were only weakly inhibitory with endogenous substrates. In contrast, diS-C3-(5) markedly inhibited the respiration of synaptosomes with glucose, pyruvate and endogenous substrates. All three probes reduced ATP content in synaptosomes and depolarized the membrane potential in synaptosomes with increasing concentrations of the probes. It is, therefore, preferable to estimate membrane potential with TPP+ or rhodamine 6G at their low concentrations where their effect on metabolism is negligible.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Brain Chemistry; Carbocyanines; Coloring Agents; Glucose; In Vitro Techniques; Lactates; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Pyruvates; Pyruvic Acid; Quinolines; Rats; Rhodamines; Succinates; Succinic Acid; Synaptosomes; Xanthenes

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiadicarbocyanine (disS-C3-(5)). The change in TPP+ concentration in the medium was measured with a TPP+-selective electrode. By monitoring differences in accumulation of TPP+ in media containing low and high potassium concentrations, a resting potential of -58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na+, K+ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP+ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP+ and diS-C3-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Membrane; Electric Stimulation; Fluorescent Dyes; Guinea Pigs; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Membrane Potentials; Neutrophils; Onium Compounds; Organophosphorus Compounds; Potassium

The protonmotive force in Saccharomyces fragilis has been estimated under various experimental conditions. The transmembrane potential has been monitored with tetraphenylphosphonium and 3,3'-dipropylthiadicarbocyanine. The distribution ratio of these cations between intracellular and extracellular water appeared to be governed by the electrical potential difference across the membrane of this yeast strain. The transmembrane pH difference was deduced from dimethyloxazolidinedione uptake experiments and from direct measurements of intracellular pH after freezing and boiling of the cells. Both methods yielded similar results. D-Fucose is transported by S. fragilis via H+ symport, with a H+/fucose stoichiometry of approximately 1. Accumulation of this sugar appeared to be closely correlated with the protonmotive force.

Topics: Benzothiazoles; Biological Transport, Active; Carbocyanines; Cell Membrane; Coloring Agents; Fucose; Hydrogen-Ion Concentration; Indicators and Reagents; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Saccharomyces

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