4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and tetraphenylphosphonium

Inside-out membrane vesicles were prepared from human red blood cells pretreated with diisothiocyano-2,2'-disulfonic stilbene to inhibit anion fluxes. The pH-sensitive probe fluorescein isothiocyanate-dextran was incorporated inside the vesicles. Formation of pH gradients due to proton transport by the sodium pump was distinguished from pH gradients formed in response to transmembrane electrical potentials generated by the pump by virtue of their insensitivity and sensitivity, respectively, to dissipation by lipophilic cations. Under the conditions used (pH 6.6), proton transport by the Na,K-ATPase was minimized, and the formation of pH gradients in response to electrical potentials was detected. Thus, the generation of a strophanthidin-sensitive, ATP-dependent electrical potential, inside positive (approximately 1 mV) upon addition of 4 meq of sodium to potassium-filled inside-out vesicles is consistent with the well documented stoichiometry of three sodium ions exchanging with two potassium ions. In contrast, when the cytoplasmic sodium concentration is reduced to less than or equal to 0.4 mM, the potential generated is of the opposite sign, i.e. inside negative, consistent with the decreased Na:K coupling ratio reported previously, i.e. Na:K(Rb) coupling ratios of approximating 1:2 when the sodium concentration is reduced to 0.2 mM (Blostein, R. (1983) J. Biol. Chem. 258, 12228-12232).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Cytoplasm; Dextrans; Erythrocyte Membrane; Erythrocytes; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Humans; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Sodium; Sodium-Potassium-Exchanging ATPase; Strophanthidin

The unidirectional influx of 36Cl- into isolated chick epithelial cells is 30% inhibited by 300 microM SITS. Characteristics of the SITS-sensitive flux pathway were examined in terms of sensitivity to changes in membrane potential and intracellular pH. Potential dependence was evaluated using unidirectional influx of [14C]tetraphenylphosphonium ([14C]-TPP+) as a qualitative sensor of diffusion potentials created by experimentally imposed gradients of Cl-. Steady-state distribution of [14C]methylamine ([14C]MA) was used to examine for Cl(-)-dependent changes in intracellular pH. Imposed Na+ gradients, but not Cl- gradients, induce changes in [14C]MA distribution. SITS does not alter the [14C]MA distribution observed in cells with imposed gradients of Na+ and Cl-. Both results suggest that inhibition of Cl(-)-OH- exchange system is not the basis for the SITS effect on Cl- influx. However, if relative permeabilities for ion pairs via conductance pathways are compared, it can be shown that SITS causes a marked reduction of Pcl relative to either PNa or PK. SITS also inhibits electrically induced influx of [14C]TPP+ or [14C] alpha-methylglucoside driven by imposed Cl- gradients. Conversely, electrically driven Cl- influx can be blocked by SITS. These observations are all consistent with a SITS-sensitive Cl- conductance pathway associated with the plasma membrane of chick intestinal cells. No Cl(-)-OH- exchange capability can be detected for chick intestinal cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Biological Transport; Cell Membrane Permeability; Chickens; Chlorides; Diffusion; Epithelium; Hydrogen-Ion Concentration; Intestinal Mucosa; Membrane Potentials; Methylamines; Onium Compounds; Organophosphorus Compounds; Quaternary Ammonium Compounds; Sodium; Stilbenes

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