Single photon radioluminescence. I. Theory and spectroscopic properties.

The excitation of a fluorescent molecule by a beta-decay electron (radioluminescence) depends upon the electron energy, the distance between radioactive 'donor' and fluorescent 'acceptor', and the excitation characteristics and solvent environment of the fluorophore. The theory for calculation of single photon radioluminescence (SPR) signals is developed here; in the accompanying paper, measurement methods and biological applications are presented. To calculate the three-dimensional spatial profile for electron energy deposition in an aqueous environment, a Monte Carlo calculation was performed incorporating theories of electron energy distributions, energy loss due to interactions with matter, and deflections in electron motion due to collisions. For low energy beta emitters, 50% of energy deposition occurs within 0.63 micron (3H, 18.5 keV), 22 microns (14C, 156 keV), 25 microns (35S, 167 keV), and 260 microns (36Cl, 712 keV) of the radioisotope. In close proximity to the beta emitter (100 nm, 3H; 10 microns, 14C) the probability for fluorophore excitation is approximately proportional to the inverse square of the distance between the beta emitter and fluorophore. To investigate the other factors that determine the probability for fluorophore excitation, SPR measurements were carried out in solutions containing 3H and a series of fluorophores in different solvents. In water, the probability of fluorescence excitation was nearly proportional to the integrated absorbance over a > 1,000-fold variation in absorbances. The probability of fluorescence excitation was enhanced up to 2,600-fold when the fluorophore was in a "scintillant" aromatic or hydrocarbon solvent. SPR emission spectra were similar to fluorescence emission spectra obtained with photon excitation. The single photon signal due to Bremsstrahlung increased with wavelength in agreement with theory. The distance dependence for the SPR signal predicted by the model was in good agreement with measurements in which a 14C donor was separated by known thicknesses of water from a fluorescently-coated coverglass. Quantitative predictions for radioluminescence signal as a function of donor-acceptor distance were developed for specific radioisotope-fluorophore geometries in biological samples.     

Distance between Cys-201 in erythrocyte band 3 and the bilayer measured by single-photon radioluminescence.

Single-photon radioluminescence (SPR), the excitation of fluorophores by short-range beta-decay electrons, was developed for the measurement of submicroscopic distances. The cytoplasmic domain of band 3 (cdb3) is the primary, multisite anchorage for the erythrocyte skeleton. To begin to define the membrane arrangement of the highly asymmetrical cdb3 structure, the distance from the bilayer of Cys-201 next to the "hinge" of cdb3 was measured by both SPR and resonance energy transfer (RET). cdb3 was labeled at Cys-201 with fluorescein maleimide. For SPR measurements, the bilayer was labeled with [3H]oleic acid. The corrected cdb3-specific SPR signal was 98 +/- 2 cps microCi-1 [mumol band 3]-1. From this and the signal from a parallel sample in which 3H2O was substituted for [3H]oleic acid to create uniform geometry between 3H and the fluorophores, a Cys-201-to-bilayer separation of 39 +/- 7 A was calculated. Confirmatory distances of 40 and 43 A were obtained by RET between fluorescein on Cys-201 and eosin and rhodamine B lipid probes, respectively. This distance indicates that Cys-201 lies near band 3's vertical axis of symmetry and that the subdomain of cdb3 between the hinge and the membrane is not significantly extended. In addition, these results validate SPR as a measure of molecular distances in biological systems.     

Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate

Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.     

Identification of an anion channel protein from electric organ of Narke japonica

The anion permeability of membrane vesicles prepared from the electric organ of $\text{Narke japonica}$ was inhibited by the addition of 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS). The permeability was measured by measuring changes in the scattered-light intensity caused by the osmotic volume change of vesicles; and also by the efflux measurement of ions from the vesicles using radioisotopes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane vesicles treated with dihydro analog of DIDS ([³H]H₂DIDS) showed that the H₂DIDS binding protein has a molecular weight of 180,000, and exists in membrane vesicles as a dimer formed by a disulfide bond between monomers of molecular weight 90,000.     

Permeation of the erythrocyte stroma by superoxide radical.

Superoxide anion, generated by xanthine oxidase within vesicles formed from washed erythrocyte ghosts, crosses the vesicle membrane to reduce cytochrome c in the medium (Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem, 253, 1838-1845). To determine whether O2- could travel through the membrane in the "channel" for the exchange of stable anions, the effects of two specific inhibitors of anion exchange, 4-acetamido-4'-isothiocyano-2,2' disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS), on the escape of O2- from vesicles were studied. The reduction of external cytochrome c, caused by O2- produced by the enzymic turnover of internal xanthine oxidase, was 85 to 90% inhibited by SITS and DIDS. If SITS impeded the egress of O2- from vesicles, it should enhance the internal effects of O2- and antagonize the inhibition of these effects by external superoxide dismutase. External superoxide dismutase inhibited the lysis of vesicles containing xanthine oxidase. SITS (60 micron) partially reversed this inhibition. It appears that O2- can cross the membrane of the erythrocyte in the anion channel.     

Activation of gamma-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a.

The interaction of avermectin B1a (AVMB1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with 36Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB1a-stimulated chloride release required the presence of intact vesicles. The efflux of 36Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB1a produced half-maximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 +/- 0.3 microM and a 35.4 +/- 1.4% maximal loss of intravesicular chloride at saturating concentrations. gamma-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB1a-stimulated chloride efflux. These data demonstrate that AVMB1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.     

Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor

Surface plasmon resonance (SPR)-based sensors have been used to detect the binding between interactive molecules. We applied the SPR technology to the analysis of interactions between living cells and molecules reactive to the cells, using mast cells and mast cell-reactive antigens. The exposure of dinitrophenol-human serum albumin (DNP-HSA), an antigen that stimulates mast cells, to IgE-sensitized mast cells induced a robust and long-lasting SPR signal in a dose-dependent manner. The maximal increase in SPR signal induced by 100 ng/ml DNP-HSA was 0.200 +/- 0.120 angle (mean +/- SD, n = 37), about 1000 times larger than the theoretically expected increase for the simple binding of DNP-HSA to Fc(epsilon)RI, the high-affinity IgE receptor. A small, but similarly prolonged signal was observed when the cells were stimulated by an agonist of the adenosine A3 receptor. The signal induced by DNP-HSA was abolished by genistein, and partially inhibited by phorbol 12-myristate 13-acetate and wortmannin. Interestingly, the signal induced by DNP-HSA was only weakly inhibited by DNP-lysine, suggesting that DNP-lysine manifests its action not by inhibiting, but by modulating the crosslinking of Fc(epsilon)RI. We concluded that SPR sensors can detect biologically significant signals in a real-time manner from the interactions between cells and molecules reactive to the cells.     

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Anion transport in relation to proteolytic dissection of band 3 protein

Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55 000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3- transporters by Ca2(+)-mobilizing agonists

Pancreatic acini loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca2(+)-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, a while in HCO3(-)-buffered medium pHi is determined by the combined activities of a Na+/H+ exchanger, a Na(+)-HCO3- cotransporter and a Cl-/HCO3- exchanger. In this study we found that stimulation of acini with Ca2(+)-mobilizing agonists in HEPES or HCO3(-)-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPES-buffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H+ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCO3(-)-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO3(-)-buffered medium: (a) recovery from acid load in the presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) (Na+/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO3- influx and efflux, respectively, by Na(+)-HCO3- cotransport) were inhibited; and (c) HCO3- influx and efflux due to Cl-/HCO3- exchange, which was measured by changing the Cl- or HCO3- gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO3- exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO3- influx due to Na(+)-HCO3- cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO3(-)-buffered medium, as much as treating the acini with H2DIDS resulted in similar agonist-mediated pHi changes in HEPES- and HCO3(-)-buffered media. The effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.     

Trimethylamine oxide transport across plasma membranes of elasmobranch erythrocytes.

The aim of this study was to characterize the erythrocyte cell membrane transport of trimethylamine oxide (TMAO) in the little skate, Raja erincea. Uptake of TMAO occurs by two processes, Na(+)-dependent and Na(+)-independent. 2,4 dinitrophenol (2,4 DNP), a known ATP synthesis inhibitor, inhibited TMAO uptake, suggesting the involvement of the Na(+)/K(+)-ATP pump in Na(+)-dependent TMAO transport. Na(+)-independent TMAO uptake was stimulated by cell swelling when erythrocytes were incubated in hypotonic elasmobranch incubation medium. Swelling-activated, Na(+)-independent TMAO uptake was inhibited by the anion transport inhibitors quinine and 4, 4'-diisthiocyanostilbene-2,2'-disulfonic acid (DIDS), two blockers of the swelling-activated osmolyte channel in skate erythrocytes. TMAO efflux was stimulated by hypotonic stress in the erythrocytes of the spiny dogfish, Squalus acanthias. DIDS also inhibited this efflux, indicating that TMAO is transported by the organic osmolyte channel in the erythrocytes of this elasmobranch as well. J. Exp. Zool. 284:605-609, 1999.     

Polarized distribution of Na+/H+ antiport and Na+/HCO3- cotransport in primary cultures of renal inner medullary collecting duct cells.

Primary cultures of rat renal inner medullary collecting duct cells were grown to confluence on glass coverslips and treated permeant supports, and the pH-sensitive fluorescent probe 2,7-biscarboxyethyl-5,6-carboxyfluorescein was employed to delineate the nature of the transport pathways that allowed for recovery from an imposed acid load in a HCO3-/CO2-buffered solution. The H+ efflux rate of acid-loaded cells was 13.44 +/- 0.94 mM/min. Addition of amiloride, 10(-4) M, to the recovery solution reduced the H+ efflux rate to 4.06 +/- 0.63 mM/min. The amiloride-resistant pHi recovery mechanism displayed an absolute requirement for Na+ but was Cl(-)-independent. Studies performed on permeable supports demonstrated that the latter pathway was located primarily on the basolateral-equivalent (BE) cell surface and was inhibited by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In a Na(+)-replete solution containing DIDS (50 microM) and amiloride (10(-4) M), acid-loaded cells failed to return to basal pHi. To delineate further the amiloride-inhibitable component of pHi recovery, monolayers were studied in the nominal absence of HCO3-/CO2. In 70% of monolayers studied, Na(+)-dependent, amiloride-inhibitable H+ efflux was the sole mechanism whereby acid-loaded cells returned to basal pHi. A Na(+)-independent pathway was observed in 30% of monolayers examined and represented only a minor component of the pHi recovery process. In studies performed on permeable supports, the Na(+)-dependent amiloride-inhibitable pathway was found to be confined exclusively to the BE cell surface. In summary, confluent monolayers of rat renal inner medullary collecting duct cells in primary culture possess two major mechanisms that contribute toward recovery from an imposed acid load, namely, Na+/H+ antiport and Na+/HCO3- cotransport. Na(+)-independent pHi recovery mechanisms represent a minor component of the pHi recovery process in the cultured cell. Both the Na+/H+ antiporter and Na+/HCO3- cotransporter are located primarily on the BE cell surface.     

Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Influence of vesicle curvature on fluorescence relaxation kinetics of fluorophores

The effect of membrane curvature on the fluorescence decay of 2-p-toluidinyl-naphthalene-6-sulfonic acid (TNS), 2-(9-anthroyloxy) stearic acid (2-AS) and 12-(9-anthroyloxy)-stearic acid (12-AS) was investigated for egg lecithin vesicles of average diameter dm = 22 nm and 250 nm. The biexponential fluorescence decay of TNS at the red edge of the emission spectrum was analysed according to the model of Gonzalo and Montoro [1]. Over the entire temperature range (1-40 degrees C) the small TNS labelled vesicles showed significantly shorter solvent relaxation times tau(r) than their larger counterparts (e.g. 1.3 ns compared with 2.1 ns at 5 degrees C), indicating a higher mobility of the hydrated headgroups in the highly curved, small vesicles. The fluorescence decay of both AS derivatives is also biexponential. While the shorter decay times (1-3 ns) are practically identical for small and large vesicles, the longer decay times (5-14 ns) are identical only for 12-AS but not for 2-AS. This indicates that the microenvironment is similar in small and large vesicles deep in the membrane in spite of the differences in curvature.     

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.     

Iodide transport in lactating rat mammary tissue via a pathway independent from the Na+/I- cotransporter: evidence for sulfate/iodide exchange

Although it is beyond doubt that mammary cells accumulate iodide via a Na+-dependent transport mechanism, it is not clear if this is the only pathway for iodide transport in mammary tissue. In view of this, experiments were designed to test for the presence of an anion-exchange pathway which could mediate the transport of iodide into mammary cells; thus, the effect of external iodide on sulfate efflux from rat mammary tissue has been investigated. Iodide trans-stimulated sulfate efflux from mammary tissue explants in a dose-dependent manner: 0.1, 1.0 and 10.0 mM iodide stimulated the fractional release of iodide by 56 +/- 2.2, 166.5 +/- 17.5, and 302.9 +/- 29.8%, respectively. The stimulation of sulfate efflux by external iodide was completely inhibited by DIDS (4.4'-diisothiocyanatostilbene 2,2'-disulfonic acid). Perchlorate (1 mM) also trans-stimulated sulfate efflux in a manner that was inhibited by DIDS. Furthermore, iodide trans-accelerated sulfate efflux from rat mammary acini via a DIDS-sensitive mechanism. The results are consistent with the presence of a DIDS-sensitive anion-exchange mechanism which can accept iodide as a substrate. It appears that the iodide-sulfate exchange mechanism is independent from the sodium-dependent iodide transporter given that sulfate is not a substrate of the latter system. The iodide-sulfate exchanger may operate in parallel with the sodium-dependent iodide transporter to mediate iodide uptake into mammary cells.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.