valinomycin and triphenylmethylphosphonium

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Chlorine; Gluconates; Glucose; Glycerol; Ionophores; Ions; Membrane Potentials; Oligomycins; Onium Compounds; Potassium; Rubidium Radioisotopes; Sodium; Sodium-Potassium-Exchanging ATPase; Time Factors; Trityl Compounds; Trypanosoma brucei brucei; Uncoupling Agents; Valinomycin

The distribution of 86Rb+ and the radiolabelled lipophilic cation [3H]methyltriphenylphosphonium (MePh3P+) was used to investigate the membrane potentials that exist in bloodstream forms of Trypanosoma brucei. Even after correction for binding to cellular constituents, the accumulation of MePh3P+ was approximately tenfold greater than the accumulation of Rb+ under resting conditions. The addition of low concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin reduced the accumulation of MePh3P+ tenfold without perturbing the accumulation of Rb+. Although selective permeabilization of the plasma membrane abolished the accumulation of Rb+ and caused a substantial decrease in the accumulation of MePh3P+, a significant carbonylcyanide-p-trifluoromethoxyphenylhydrazone-sensitive accumulation of MePh3P+ persisted under these conditions. These data were consistent with the presence of at least two distinct membrane potentials (delta psi) in bloodstream forms of T. brucei; a potential across the plasma membrane (delta psi p) and an additional delta psi, generated by the electrogenic movement of H+, across the membrane of an intracellular organelle that possesses no electrical permeability to Rb+ or K+.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane Permeability; Indicators and Reagents; Membrane Potentials; Onium Compounds; Osmolar Concentration; Rats; Rubidium; Trityl Compounds; Trypanosoma brucei brucei; Valinomycin

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.

Topics: Adenosine Triphosphate; Animals; Cell Membrane; Chlorides; Chlorine; Female; Indicators and Reagents; Kinetics; Liver; Membrane Potentials; Onium Compounds; Potassium; Radioisotopes; Rats; Rats, Inbred Strains; Rhodamines; Rubidium Radioisotopes; Staining and Labeling; Trityl Compounds; Trypan Blue; Valinomycin

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cells, Cultured; Cytoplasm; Drug Interactions; Energy Metabolism; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Liver; Male; Membrane Potentials; Mitochondria, Liver; Monensin; Oligomycins; Onium Compounds; Potassium Cyanide; Rats; Rats, Inbred Strains; Trityl Compounds; Valinomycin

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Topics: Adenine Nucleotides; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Energy Metabolism; Gluconeogenesis; Indicators and Reagents; Intracellular Membranes; Liver; Male; Membrane Potentials; Mitochondria, Liver; Oligomycins; Onium Compounds; Rats; Rats, Inbred Strains; Staining and Labeling; Thermodynamics; Trityl Compounds; Trypan Blue; Urea; 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

A Cl--stimulated adenosinetriphosphatase (ATPase) activity and an ATP-dependent Cl- transport process were found in Aplysia enterocyte plasma membranes. In an attempt to further elucidate this transport process plasma membrane vesicles from Aplysia enterocytes were prepared utilizing differential centrifugation and sucrose density gradient techniques. Electrogenicity of the ATP-dependent Cl- transport was confirmed in three ways. First, an inwardly directed valinomycin-induced K+ diffusion potential, making the vesicle interior electrically positive, enhanced ATP-driven Cl- uptake compared with vesicles lacking the ionophore. Second, ATP plus Cl- increased intravesicular negativity measured by lipophilic triphenylmethylphosphonium distribution across the vesicular membrane. Third, both vanadate and thiocyanate inhibited the ATP plus Cl--dependent intravesicular negativity. These results are consistent with the hypothesis that the active electrogenic Cl- transport mechanism in Aplysia intestine could be a Cl--stimulated ATPase found in the enterocyte plasma membrane.

Topics: Adenosine Triphosphate; Algorithms; Animals; Aplysia; Biological Transport, Active; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Chlorides; Electrophysiology; Intestinal Mucosa; Intestines; Membrane Potentials; Onium Compounds; Potassium; Reference Values; Trityl Compounds; Valinomycin

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.

Topics: Biological Transport; Carbodiimides; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Dicyclohexylcarbodiimide; Halobacterium; Hydrogen-Ion Concentration; Light; Onium Compounds; Organotin Compounds; Sodium; Trityl Compounds; Valinomycin

The rates of oxidation and phosphorylation in isolated rat-liver mitochondria have a steep dependence on the protonmotive force (delta mu H+) across the membrane. These experimentally observed relationships proved to be independent of the way in which delta mu H+ was varied. These results were obtained when the membrane potential (delta psi) was calculated from the distribution of K+ (in the presence of valinomycin). When triphenylmethylphosphonium (TPMP+) was used as a probe for delta psi, slightly different flow-force relationships were obtained. We conclude that unique relationships exist between delta mu H+ and the rates of oxidation and phosphorylation, and that under some conditions the behaviour of the probe TPMP+ is anomalous.

Topics: Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Mitochondria, Liver; Onium Compounds; Oxidative Phosphorylation; Oxygen Consumption; Potassium; Rats; Trityl Compounds; Valinomycin

A rapid cellular-fractionation technique [ Hoek , Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.

Topics: Animals; Cell Fractionation; Digitonin; Intracellular Membranes; Kinetics; Liver; Male; Membrane Potentials; Mitochondria, Liver; Onium Compounds; Rats; Rats, Inbred Strains; Trityl Compounds; Valinomycin

A heterotrophic nitrifying Alcaligenes sp. from soil was grown as a denitrifier on nitrate and subjected to oxidant pulse experiments to ascertain the apparent efficiencies of proton translocations during O2 and nitrogen-oxide respirations. With endogenous substrate as the reducing agent the leads to H+/2e- ratios, extrapolated to zero amount of oxidant per pulse, were 9.4, 3.7, 4.3 and 3.5 for O2, nitrate, nitrite and N2O, respectively. The value for O2 and those for the N-oxides are, respectively, somewhat larger and smaller than corresponding values for Paracoccus denitrificans. None of the three permeant ions employed with the Alcaligenes sp. (valinomycin-K+, thiocyanate and triphenylmethylphosphonium) was ideal for all purposes. Thiocyanate provided highest ratios for O2 but abolished the oxidant pulse response for nitrate and N2O. Valinomycin was slow to penetrate to the cytoplasmic membrane and relatively high concentrations were required for optimal performance. Triphenylmethylphosphonium enhanced passive proton permeability and diminished proton translocation at concentrations required to realize the maximal oxidant pulse response.

Topics: Alcaligenes; Hydrogen; Nitrates; Nitrites; Nitrogen; Nitrogen Oxides; Onium Compounds; Oxygen Consumption; Protons; Thiocyanates; Trityl Compounds; Valinomycin

Unidirectional fluxes of triphenylmethylphosphonium and of Cs+ as its valinomycin complex were studied using trace concentrations of the cations. The rate constants of influx and efflux were estimated mainly at 0 degrees C from the uptake kinetics in respiring mitochondria and the in/out ratios in the steady state. The efflux rate constants in the energized state were also measured after dilution of the mitochondrial suspension in the steady state, and in deenergized mitochondria from the efflux rates of cations after inhibition of respiration. It was found that the energy state of mitochondria had little effect on the rate constants of efflux, while the rate of influx was strongly stimulated by respiration. The former finding is not readily explained by the classical chemiosmotic theory, since a transmembrane potential, negative on the inside, formed on energization would be expected to strongly inhibit the efflux of cations. The data may be explained by a pump-and-leak model in which localized electrical fields in hydrophobic domains of the membrane are coupled to the pumping of hydrophobic cations against an electrochemical gradient, while leaks would effect efflux.

Topics: Animals; Biological Transport; Cesium; Indicators and Reagents; Kinetics; Mathematics; Mitochondria, Liver; Models, Biological; Onium Compounds; Rats; Trityl Compounds; Valinomycin

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52-60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

Topics: Benzothiazoles; Blood Platelets; Carbocyanines; Electrolytes; Humans; Inulin; Membrane Potentials; Onium Compounds; Platelet Aggregation; Spectrometry, Fluorescence; Trityl Compounds; Valinomycin

Topics: Animals; Cell Membrane; Lymphocytes; Membrane Potentials; Mitochondria; Onium Compounds; Potassium; Rubidium; Swine; Trityl Compounds; Valinomycin

Topics: Anti-Bacterial Agents; Cesium; Macrolides; Membrane Potentials; Mitochondria; Onium Compounds; Rotenone; Rubidium; Succinates; Thallium; Trityl Compounds; Valinomycin

Isolated synaptic plasma membrane vesicles developed an internal negative membrane potential (delta psi) following loading with potassium succinate and incubation in NaCl, sodium succinate, or Tris succinate media. Membrane delta psi was monitored by measuring triphenyl[3H]methylphosphonium ion ([3H]TPMP+) accumulation by these vesicles. Estimates of delta psi ranged from --6.9 mV for vesicles incubated in sodium succinate to --28 mV for membranes incubated in NaCl. Intravesicular TPMP+ accumulation was strongly dependent on the K+ diffusion potential and was enhanced by the K+ ionophore valinomycin and by the adenosine analog 2-chloroadenosine (2-Cl-Ado). The stimulation of TPMP+ influx by 2-Cl-Ado was dependent on the concentration of this agent, independent of Cl- fluxes, and sensitive to inhibition by the methylxanthine theophylline. The increase of delta psi of the synaptic membrane vesicles caused by 2-Cl-Ado paralleled the hyperpolarization of neurons produced by adenosine and 2-Cl-Ado in physiological systems.

Topics: 2-Chloroadenosine; Adenosine; Animals; Brain; Membrane Potentials; Onium Compounds; Potassium; Rats; Sodium Chloride; Succinates; Succinic Acid; Synaptic Membranes; Trityl Compounds; Valinomycin

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin

The accumulation of the lipophilic cation, triphenylmethylphosphonium, has been employed to determine the resting membrane potential in human erythrocytes, turkey erythrocytes, and rat white adipocytes. The triphenylmethylphosphonium cation equilibrates rapidly in human erythrocytes in the presence of low concentrations of the hydrophobic anion, tetraphenylborate. Tetraphenylborate does not accelerate the uptake of triphenylmethylphosphonium ion by adipocytes. The cell associated vs. extracellular distribution of the triphenylmethylphosphonium ion is proportional to changes in membrane potential. The distribution of this ion reflects the membrane potential determining concentration of the ion with dominant permeability in a "Nernst" fashion. The resting membrane potentials for the human erythrocyte, turkey erythrocyte, and rat white adipocyte were found to be -8.4 +/- 1.3, -16.8 +/- 1.1, and -58.3 +/- 5.0 mV, respectively, values which compare favorably with values obtained by other methods. In addition, changes in membrane potential can be assessed by following triphenylmethylphosphonium uptake without determining the intracellular water space. The method has been successfully applied to a study of hormonally induced changes in membrane potential of rat white adipocytes.

Topics: Adipose Tissue; Animals; Calcimycin; Cations, Monovalent; Cell Membrane; Cell Membrane Permeability; Erythrocytes; Humans; Kinetics; Male; Mathematics; Membrane Potentials; Onium Compounds; Rats; Tetraphenylborate; Trityl Compounds; Turkeys; Valinomycin

Topics: Adenosine Triphosphate; Animals; Anti-Bacterial Agents; Biological Transport, Active; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cattle; Chromaffin Granules; Chromaffin System; Kinetics; Magnesium; Membrane Potentials; Onium Compounds; Trityl Compounds; Valinomycin

Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. Addition of valinomycin induces a membrane potential of approximately 50 mV (interior negative) as estimated by uptake of the lipophilic cation triphenylmethylphosphonium, but has surprisingly little effect on 5-hydroxytryptamine transport. Addition of 2,4-dinitrophenol dissipates the valinomycin-induced membrane potential. In the absence of valinomycin, 2,4-dinitrophenol has no effect on 5-hydroxytryptamine transport but valinomycin and 2,4-dinitrophenol together inhibit transport, probably by dissipation of the K+ gradient. These results are consistent with an electroneutral mechanism in which 5-hydroxytryptamine influx is directly coupled to potassium ion efflux and argue against an electrogenic mechanism in which there is a net influx of positive charge with 5-hydroxytryptamine.

Topics: Animals; Biological Transport, Active; Blood Platelets; Dinitrophenols; Kinetics; Membrane Potentials; Onium Compounds; Potassium; Serotonin; Swine; Trityl Compounds; Valinomycin

The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.

Topics: Biological Transport, Active; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Dimethadione; Halobacterium; Hydrogen-Ion Concentration; Kinetics; Light; Mathematics; Membrane Potentials; Onium Compounds; Sorbitol; Species Specificity; Sucrose; Trityl Compounds; Valinomycin