valinomycin and gluconic-acid

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

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10 microM) increased furosemide-insensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanide-insensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloride-depleted cells. When cells with normal chloride content were depolarized (135 mM medium potassium + 10 microM valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl- approximately Br- > NO3- > I-. The electrogenic transport pathway was inhibited in chloride-depleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

Topics: Anions; Biological Transport; Bumetanide; Carrier Proteins; Cations; Cell Membrane Permeability; Cell Polarity; Cell Size; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Furosemide; Gluconates; Humans; Intestinal Mucosa; Intracellular Fluid; Ionomycin; Nitrates; Organ Specificity; Quinolinium Compounds; Sodium-Potassium-Chloride Symporters; Tumor Cells, Cultured; Valinomycin

Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.

Topics: Animals; Biological Transport; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Gluconates; In Vitro Techniques; Kidney Cortex; Membrane Potentials; Microvilli; Osmolar Concentration; Phosphates; Rats; Sodium Chloride; Thermodynamics; Thiocyanates; Valinomycin