valinomycin and thiocyanate

Inhibition of gastric acid secretion by thiocyanate is explained by a protonophoric mechanism assuming that thiocyanate induces a H(+) back flux from the acidic gastric lumen into the parietal cells of gastric mucosa. Protonophoric activity of thiocyanate was examined by swelling measurements using rat liver mitochondria and theoretically by quantum chemical methods. Mitochondria suspended in K-thiocyanate medium plus nigericin (an H/K-exchanger) swelled when the medium pH was acidic, indicating that SCN(-) initiates a transfer of H(+) across the inner membrane. To rationalize the protonophoric activity of thiocyanate, we considered the dehydration of SCN(-) to be critical for transmembranal H(+) transfer. For modeling this process, various hydrate clusters of SCN(-) and Cl(-) were generated and optimized by density functional theory (DFT) at the B3-LYP/6-311++G(d,p) level. The cluster hydration energy was lower for SCN(-) than for Cl(-). The total Gibbs free energies of hydration of the ions were estimated by a hybrid supermolecule-continuum approach based on DFT. The calculated hydration energies also led to the conclusion that SCN(-) is less efficiently solvated than Cl(-). Due to the easier removal of the hydration shell of SCN(-) relative to Cl(-), SCN(-) is favored in going across the membrane, giving rise to the protonophoric activity.

Topics: Adenosine Triphosphate; Animals; Anions; Cell Membrane; Cytosol; Female; Gastric Acid; Gastric Mucosa; H(+)-K(+)-Exchanging ATPase; Hydrochloric Acid; Hydrogen; Hydrogen Bonding; Hydrogen-Ion Concentration; Ionophores; Ions; Isothiocyanates; Light; Liver; Membrane Potentials; Mitochondria, Liver; Models, Chemical; Models, Molecular; Nigericin; Parietal Cells, Gastric; Protons; Quantum Theory; Rats; Rats, Wistar; Scattering, Radiation; Temperature; Thermodynamics; Thiocyanates; Time Factors; Valinomycin; Water

An iodide channel has been previously identified in the plasma membrane of bovine throcytes [Golstein et al., Am. J. Physiol. 263 (Cell Physiol. 32): C590-C597, 1992]. The plasma membrane proteins were solubilized and ultrafiltered, and the protein fraction collected above 100 kDa was inserted in liposomes. Voltage-sensitive uptake of radiolabeled I- by these proteoliposomes was studied. To this end, an outward I- gradient was set up by loading the proteoliposomes with KI and removing extraliposomal I-. I- exit from the proteoliposome induces an inside positive membrane potential, which leads to the uptake of 125I- added to the incubation medium. This uptake was abolished by valinomycin, which in the presence of K+ short circuits the liposomal membrane potential, demonstrating the conductive nature of this uptake. A double reciprocal plot of I- influx over I- concentration suggests the existence of a single population of channels in these proteoliposomes with a Michaelis-Menten constant for I- of approximately 9 microM. When the proteoliposomes were loaded with KCl or KSCN instead of I-, no conductive uptake occurred anymore, suggesting that these anions are unable to diffuse through the I- conductance, hence do not generate a diffusion potential. I- uptake by KI-loaded proteoliposomes was not inhibited in the presence of a 1,000-fold excess of extraliposomal Cl- but was completely inhibited by a 1,000-fold excess of extraliposomal SCN-, indicating that Cl- does not permeate the I- channel, whereas SCN- inhibits it. SCN- and flufenamate were both shown to be competitive inhibitors of the I- channel with an inhibitor constant of approximately 10 and 750 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anions; Cattle; Electric Conductivity; Flufenamic Acid; Iodides; Ion Channels; Liposomes; Permeability; Proteolipids; Thiocyanates; Thyroid Gland; Valinomycin

We examined the mechanism of prostaglandin E2 transport in rabbit renal basolateral membrane vesicles which were predominantly oriented right-side-out. In the presence of an inwardly directed H+ gradient, the initial rate of uptake was markedly accelerated and the influx of prostaglandin E2 resulted in a transient accumulation (overshoot) above the equilibrium value. Both H+-independent and H+-stimulated prostaglandin E2 uptake were shown to be insensitive to valinomycin-induced K+ diffusion potentials. Intravesicular probenecid inhibited the pH gradient-stimulated uptake of prostaglandin E2 but did not affect the pH-stimulated uptake of thiocyanate and acetate which enter membranes via ionic and nonionic diffusion, respectively. Finally, the existence of a Na+ cotransport or of a K+ antiport pathway for prostaglandin E2 could not be demonstrated. Thus, these data demonstrate the presence of an electrically neutral H+-prostaglandin E2 cotransport or OH- -prostaglandin E2 antiport mechanism in the basolateral membrane of the rabbit proximal tubule.

Topics: Acetates; Acetic Acid; Animals; Basement Membrane; Biological Transport, Active; Diffusion; Dinoprostone; Kidney; Potassium; Prostaglandins E; Rabbits; Sodium; Thiocyanates; 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

The effects of thiocyanate anion (SCN-) on proton accumulation and ATP hydrolysis by hog gastric microsomal vesicles have been investigated. The addition of SCN- to vesicles in the presence of KC1 and valinomycin reduced ATP-dependent proton accumulation in a dose-dependent manner. Inhibition was most pronounced in the presence of internal SCN-, which was obtained by preincubation of vesicles with SCN- or by the addition of K+-valinomycin, which facilitates entry of SCN-. SCN- does not appear to act by inhibition of the vesicular H+ pump because 1) there were minimal effects of SCN- on the rate of ATP hydrolysis, and 2) the initial rate of pH gradient formation was greater with 20 mM SCN- than with 20 mM Cl-. In the presence of K+ and valinomycin, external SCN- inhibited ATP-dependent pH gradient formation by increasing the rate of proton efflux. Preformed pH gradients (acid interior) were rapidly dissipated by internal, but not external, SCN-. These results suggest that SCN- acts to increase the rate of passive proton loss from the vesicle interior and do not support a direct inhibition of ATP-dependent H+ translocation. The results are consistent with the formation of the permeant hydrothiocyanic acid within the vesicles, increasing the rate of proton loss. The data also lend support to the HSCN backflux hypothesis of Sanders et al. [Am. J. Physiol. 234 (Endocrinol. Metab. Gastrointest. Physiol. 3): E120-E128, 1978] for inhibition of gastric acid secretion by SCN-.

Topics: Adenosine Triphosphate; Animals; Gastric Mucosa; Hydrogen-Ion Concentration; Kinetics; Microsomes; Swine; Thiocyanates; Valinomycin; Zinc

An electrochemical potential difference for H+ was established across the plasma membrane of the anaerobe Streptococcus lactis by addition of sulfuric acid to cells suspended in potassium phosphate at pH 8 along with valinomycin or permeant anions. Subsequent acidification of the cell was measured by the distribution of salicyclic acid. A comparison between cells treated or untreated with the inhibitor N,N'-dicyclohexylcarbodiimide was used to reveal that portion of net proton entry attributable to a direct coupling between H+ inflow and synthesis of ATP catalyzed by the reversible proton-translocating ATPase of this microorganism. When the imposed electrochemical proton gradient was below 180-190 mV, proton entry was at the rate expected of passive flux, for both control cells and cells treated with the ATPase inhibitor, However, at higher driving force acidification of control cells was markedly accelerated, coincident with ATP synthesis, while acidification of cells treated with the inhibitor continued at the rate characteristic of passive inflow. This observed threshold (180-190 mV) was identified as the reversal potential for this H+ "pump". Parallel measurements showed that the free energy of hydrolysis for ATP in these washed cells was 8.4 kcal/mole (370mV). The comparison between the reversal (threshold) potential and the free energy of hydrolysis for ATP indicates a stoichiometry of 2 H+/ATP for the coupling of proton movements to ATP formation in bacteria.

Topics: Adenosine Triphosphate; Anions; Dicyclohexylcarbodiimide; Hydrogen-Ion Concentration; Lactococcus lactis; Membrane Potentials; Protons; Thiocyanates; Valinomycin