4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 3-3--dipropyl-2-2--thiadicarbocyanine

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Benzothiazoles; Body Water; Carbocyanines; Chlorides; Erythrocytes; Ethoxzolamide; Hemoglobins; Humans; Hydroxides; In Vitro Techniques; Indicators and Reagents; Light; Membrane Potentials; Osmolar Concentration; Potassium; Protons; Saline Solution, Hypertonic; Scattering, Radiation; Sodium; Sucrose

We examined whether swelling-activated K-Cl cotransport is electrogenic in human erythrocytes. Baseline membrane potential, measured by the change in fluorescence of the carbocyanine dye diS-C3-5, was not different in hypotonically swollen (-7.6 mV) or isosmotically swollen cells (-9.5 mV). We used hemisodium, a new highly selective Na ionophore, in varying concentrations, in the presence of a fixed outwardly directed Na gradient (intracellular Na, 75 mM; external Na, 1 mM) to vary membrane potential over a wide range despite identical K and Cl concentrations. The membrane potential varied between -8 and -90 mV. K influx increased slightly with hyperpolarization in swollen and nonswollen cells. However, the difference between the two fluxes, swelling-activated K influx, a measure of K-Cl cotransport, was unaffected by voltage changes, as was swelling-activated K efflux. We conclude that K-Cl cotransport in human erythrocytes is electroneutral and by inference has a 1:1 stoichiometry.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Benzothiazoles; Biological Transport; Calibration; Carbocyanines; Erythrocytes; Fluorescent Dyes; Hematocrit; Homeostasis; Humans; Ionophores; Membrane Potentials; Potassium; Potassium Chloride; Sodium

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Benzothiazoles; Carbocyanines; Chlorides; Fluorescent Dyes; Ion Channels; Kidney Medulla; Membrane Potentials; Membranes; ortho-Aminobenzoates; Swine; Valinomycin

The pH sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide were used to evaluate the Cl-/HCO-3 antiporter and proton and potassium conductances, respectively, in human red blood cell ghosts. Acidic, chloride-loaded ghosts alkalinized rapidly in pH 8.5 chloride-free media. Alkalinization could not be ascribed to conductive proton efflux with either depolarizing potassium influx or chloride efflux. Alkalinization was consequent to flux on the Cl-/HCO-3 antiporter: this process displayed saturation kinetics, competitive inhibition by external chloride, and inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The mean Kms for internal chloride, external bicarbonate, and external chloride were 2.19, 0.24, and 0.44 mM, respectively. These studies confirm both the asymmetry of this carrier and the high affinity for external HCO-3; however, the affinities for internal and external chloride are significantly greater than prior estimates. The Km for internal chloride (2.19 mM) was considerably lower than levels previously reported (20-65 mM) unless external (trans) chloride was raised above 2 mM. The present studies thus demonstrate and emphasize the critical importance of trans substrate concentration in assessing the kinetics of a carrier whose mobility is faster in the loaded than in the unloaded state.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acridine Orange; Alkalies; Benzothiazoles; Bicarbonates; Buffers; Calibration; Carbocyanines; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Kinetics; Protons; Quinolines

The characterization of the anion transport system in sarcoplasmic reticulum (SR) vesicles was performed by kinetic analysis of the reversible inhibition of gluconate efflux by a disulfonic stilbene derivative; 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate (SITS). The gluconate efflux in SR vesicles was measured by following the change in Cl- ion-diffusion potential due to the mutual diffusion of Cl- and gluconate- using a potential probe; 3,3'-dipropylthiadicarbocyanine iodide (diS-C3-(5)). The main results are as follows. (1) Gluconate efflux was increased by intravesicular gluconate- and was decreased by both extravesicular gluconate- and extravesicular Cl-. (2) Gluconate efflux was depressed when membrane potential became inside-negative. (3) SITS reacted with the site of the anion transport system from the outside of the vesicles and one molecule of SITS inhibited each transport unit. (4) Extravesicular Cl- competed with SITS at the common site of the anion transport system. The dissociation constants for Cl- and SITS are 70 mM and 0.048 microM, respectively. (5) The inhibition of gluconate efflux by SITS was noncompetitive with both intravesicular gluconate and extravesicular gluconate. These results suggest that the anion transport system in SR vesicles can be described by a carrier-mediated transport model in which anions are transported by a mobile transport site accompanied by a net negative charge.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anions; Benzothiazoles; Biological Transport; Carbocyanines; Chlorides; Diffusion; Dose-Response Relationship, Drug; Fluorescent Dyes; Gluconates; Kinetics; Membrane Potentials; Quinolines; Rabbits; Sarcoplasmic Reticulum; Stilbenes