Regulation of the calcium release channel from skeletal muscle by suramin and the disulfonated stilbene derivatives DIDS,DBDS, and DNDS

Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer composition. Replacement of neutral lipids by negative phosphatidylserine increased K(a) fourfold, suggesting that reversible binding sites are near the bilayer surface. Suramin and the stilbene derivatives adsorbed to neutral bilayers with maximal mole fractions between 1-8% and with affinities approximately 100 micro M but did not adsorb to negative lipids. DIDS activated RyRs by two nonreversible mechanisms, distinguishable by their disparate DIDS binding rates (10(5) and 60 M(-1) s(-1)) and actions. Both mechanisms activated RyRs via several jumps in open probability, indicating several DIDS binding events. The fast and slow mechanisms are independent of each other, the reversible mechanism and ATP binding. The fast mechanism confers DIDS sensitivity approximately 1000-fold greater than previously reported, increases Ca(2+) activation and increases K(i) for Ca(2+)/Mg(2+) inhibition 10-fold. The slow mechanism activates RyRs in the absence of Ca(2+) and ATP, increases ATP activation without altering K(a), and slightly increases activity at pH < 6.5. These findings explain how different types of DIDS activation are observed under different conditions.     

Stilbene disulfonates block ATP-sensitive K+ channels in guinea pig ventricular myocytes

Effects of stilbene disulfonates on single K_ATP channel currents were investigated in inside-out and outside-out membrane patches from guinea pig ventricular myocytes. All drugs tested, 4,4′-diisothiocyanatostilbene, 2,2′-disulfonic acid (DIDS), 4-acetamido0-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), and 4,4′-diaminostilbene-2,2′-disulfonic acid (DADS), inhibited the K_ATP channel when they were applied to the intracellular, but not extracellular side of the membrane patch. Inhibitory actions of DIDS and SITS were irreversible, whereas those induced by DNDS and DADS were reversible. K_ATP channel inhibition was concentration dependent with an order of potency of DIDS>SITS ≈ DNDS > DADS; the Hill coefficient was close to unity for each drug. No change in channel conductance was observed during exposure to DIDS or DNDS; however, channel kinetics was altered. Distribution of the open time within bursts and that between bursts could be described by a single exponential relation in the absence and presence of DIDS or DNDS. The time constant of the open time within bursts was not altered, but that between bursts was decreased by DIDS (from 40.0±8.1 to 29.8±6.7 msec, P< 0.05) and by DNDS (from 43.1±9.3 to 31.9±7.1 msec, P<0.05). Distributions of closed time within bursts were also fitted to a single exponential function both in the absence and presence of drugs, while those of the closed time between bursts were fitted to a single exponential function in the absence of drugs, but a double exponential function was required in the presence of drugs. The rates of onset and development of channel inhibition by DIDS and DNDS appeared to be concentration dependent; a longer time was required to reach a new steady-state of channel activity as drug concentration was decreased. Inhibition by DIDS or DNDS was regulated by intracellular pH; inhibition was greater during acidic conditions. For DIDS (0.1 mm), the open probability (P _o) expressed as a fraction of the value before drug application was 42.9±8.3% at pH 7.4 and 8.2±6.6% at pH 6.5 (P<0.01); corresponding values for DNDS (1 mm) were 39.6±17.6 and 8.9 ±5.8%, respectively (P<0.01). From these data, we conclude that stilbene disulfonates block the K_ATP channel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation in an “intermediate lipid compartment” between the cytosolic drug and the site of drug action.     

Modification of K conductance of the squid axon membrane by SITS

The effects of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) on the K conductance, gK, were studied in internally perfused giant axons from squid, Doryteuthis. SITS at 3-200 microM was applied intracellularly by adding the reagent to the internal perfusion fluid. Three remarkable changes in gK were noted: there was a slowing of the opening and closing rates of the K channel in the whole voltage region; K channels modified with SITS started to open at voltages below -100 mV, and thus 30% of total K channels were open at the level of normal resting potential (approximately -60 mV) after the maximal drug effect was attained (less than 30 microM); there was a disappearance of gK inactivation that became distinct at relatively high temperature (greater than 8 degrees C). These drug effects depended solely on the drug concentration, not on factors such as repetitive alterations of the membrane potential, and the changes in gK were almost irreversible. Another disulfonic stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), had similar effects on gK, but the effects were approximately 1.5 times stronger. These changes in gK were somewhat similar to alterations in gNa produced by an application of veratridine, batrachotoxin, and grayanotoxin, which are known as Na channel openers.     

Effect of anion transport blockers on CFTR in the human sweat duct

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.     

Modulation and block of the plasma membrane anion channel of guard cells by stilbene derivatives

An anion channel in the plasma membrane of guard cells (GCAC1) provides a regulatory element for the voltage-dependent release of anions during stomatal closure (Keller et al. 1989) as well as excitability (Hedrich et al. 1990). Recognition sites for plant growth hormones on the extracellular surface of GCAC1 further indicate that this channel may also serve as a transduction element in hormone signaling (Marten et al. 1991 a). Stilbene derivatives were used to study the inhibitor-structure channel-function relationship of GCAC1. We have analyzed the activity, voltage-gate and kinetics of this channel as affected by stilbenes. The stilbene derivatives SITS and DNDS caused a shift in activation potential and a decrease in the peak current amplitude. Channel block through the action of DIDS, on the other hand, was not accompanied by a shift in voltage-dependence. Differences in the dose-dependence of the two effects give clues to the presence of channel sites responsible for gate-shifting and block. The ability to inhibit anion currents (K_d) increased in the sequence: SITS (4 µM) < DNDS (0.5 µM) < DIDS (0.2 µM). All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion-channels is characterized by a reduction of long open-transitions into flickering bursts and a decrease in channel amplitude.Abbreviations DIDS 4,4-Diisothiocyanostilbene-2,2-disulfonic acid - SITS 4-Acetamido-4-isothiocyanostilbene-2,2-disulfonic acid - DNDS 4,4-dinitrostilbene-2,2-disulfonic acid - NPPB 5-Nitro-2-(3-phenylpropylamio)benzoic acid - IAA-94 [(6,7-Dichloro2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5y1)oxy] acetic acid - A-9-C Anthracene-9-carboxylic acid - TEA Tetraethylammonium     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Stilbene disulfonic acids. CD4 antagonists that block human immunodeficiency virus type-1 growth at multiple stages of the virus life cycle

The stilbene disulfonic acids 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid and, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid bound the variable-1 immunoglobulin-like domain of CD4 on JM cells. The interaction blocked the binding of the anti-CD4 monoclonal antibody OKT4A and the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1). DIDS inhibited the acute infection of CD4+ cells by HIV-1 with a potency (IC50 approximately 30 microM) similar to that which blocked gp120 binding (IC50 approximately 20 microM) to the cellular antigen. Pretreating uninfected CD4+ C8166 cells with DIDS blocked their fusion with chronically infected gp120+ cells. DIDS covalently and selectively modified lysine 90 of soluble CD4 and abolished the gp120-binding and antiviral properties of the recombinant protein. When added to cells productively infected with HIV-1, DIDS blocked virus growth and cleared cultures of syncytia without inhibiting cellular proliferation. The stilbene disulfonic acids are a novel class of site-specific CD4 antagonists that block multiple CD4-dependent events associated with acute and established HIV-1 infections.     

Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum

Ca2+ channels of isolated sarcoplasmic reticulum were incorporated into a planar lipid bilayer and their pharmacological properties were studied. The results show that the channel is a Ca2+-induced Ca2+ release channel like that observed in skinned muscle fibers and isolated vesicles. (i) The open channel probability was increased by the addition of micromolar amounts of Ca2+ to the cis (myoplasmic) side and further increased by millimolar ATP. (ii) The channel was closed by millimolar Mg2+ and micromolar ruthenium red. We found that two disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), when added to the cis side open the channel and lock it irreversibly at open without changing the single channel conductance. Ca2+ efflux from SR vesicles was also enhanced by SITS and DIDS, as monitored by a tracer assay. Further, Ag+ activated the channel transiently. These results suggest that certain amino and SH residues play important roles in gating the Ca2+ channel.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

A systematic study was made of the action of 4-acet-amido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer 45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS, Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton.     

4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid inhibits CD3-T cell antigen receptor-stimulated Ca2+ influx in human T lymphocytes

Stimulation of the CD3-T cell antigen receptor complex on T lymphocytes results in a rapid rise in intracellular calcium from both intra- and extracellular sources. The former is thought to be released from the endoplasmic reticulum in response to inositol trisphosphate, while the latter enters the cells through a membrane potential-sensitive transporter (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590). In this report we show that the stilbene disulfonate, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), inhibited the ability of monoclonal anti-CD3 complex antibodies to stimulate an influx of calcium in the human T lymphocyte cell line, Jurkat. DIDS had no effect on either antibody binding to the receptor or receptor-stimulated phosphatidylinositol turnover. The Ki was approximately 25 microM in the presence of extracellular Cl- and 10 microM when labeling was performed in the absence of Cl-, suggesting that DIDS was competing with Cl- for binding to the cell membrane. The reduced form of DIDS, dihydroDIDS, was only 50% as effective as DIDS itself, and the monoisothiocyanate stilbene, 4-acetamido-4'-isothiocyantostilbene-2,2'-disulfonic acid, was totally ineffective, even to concentrations of 0.750 mM. Removal of extracellular Cl- also inhibited the antibody-stimulated influx of calcium. These data suggest that the function of the CD3-T cell receptor-activated calcium channel/transporter may be dependent on or regulated by extracellular Cl-.     

Blockers of VacA provide insights into the structure of the pore

The cytotoxic effects of the Helicobacter pylori toxin VacA, an important etiogenic factor in human gastric diseases, are due to its ability to form anion-selective pores in target cell membranes. We have studied the inhibition of channel activity by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), representatives of two popular classes of chloride channel blockers, to gain information on the mechanism of blocking and on the unknown structure of the VacA pore. The data indicate that both compounds produce a fast block by binding to separate but mutually exclusive sites within the channel lumen. DIDS binds close to the pore opening on the side of protein insertion, whereas NPPB blocks at a position in the opposite half of the channel. Although DIDS reaches the blocking site by traveling along the lumen, inhibition by NPPB appears to involve mainly partition of the compound into the membrane, voltage-independent diffusion from it to the inhibitory position, and voltage-dependent exit. The data are consistent with a pore that can be more easily entered from the side of protein insertion than from the opposite end.     

Influence of ion channels on the proliferation of human chondrocytes

The goal of the study was to examine connections between ion channel activity and the proliferation of human chondrocytes. Chondrocytes were isolated form human osteoarthritic knee joint cartilage. In this study the concentration-dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-aminopyridine (4-AP), 4',4' diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), verapamil (vp) and lidocaine (lido) on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry and the measurement of (3)H-thymidine incorporation as measure for the cell proliferation. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The maximal measurable effects of the membrane potential were listed with 0.25 mmol/l verapamil (-18%) and 0.1 mmol/l lidocaine (+20%). When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. After 12 days culture duration, lidocaine and 4-AP cause an increase of the DNA synthesis rate being a limited effect. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation. To gain knowledge of the regulation of chondrocyte proliferation via ion channel modulators could serve the research of new osteoarthritis treatment concepts.     

Voltage-dependent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives

When giant axons of squid, Sepioteuthis, were bathed in a 100 mM Ca-salt solution containing tetrodotoxin (TTX) and internally perfused with a solution of 100 mM tetraethylammonium-salt (TEA-salt) or tetramethylammonium-salt (TMA-salt), the membrane potential was found to become sensitive to anions, especially Cl-. Membrane currents recorded from those axons showed practically no time-dependent properties, but they had a strong voltage-dependent characteristic, i.e., outward rectification. Cl- had a strong effect upon the voltage-dependent membrane currents. The nonlinear property of the currents was almost completely suppressed by some disulfonic stilbene derivatives applied intracellularly, such as 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are blockers of chloride transport. On the basis of these experimental results, it is concluded that a voltage-dependent chloride-permeable channel exists in the squid axon membrane. The chloride permeability (PCl) is a function of voltage, and its value at the resting membrane (Em = -60 mV) is calculated, using the Goldman-Hodgkin-Katz equation, to be 3.0 X 10(-7) cm/s.     

Preparation and characterization of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and related stilbene disulfonates

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 1H and 13C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also decomposed in solution, while disodium 4,4'-dinitrostilbene-2,2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.     

Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.

AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonate) and DNDS (4,4'-dinitrostilbene-2,2'-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes upon noncovalent binding of DNDS. Since conformational changes induced by stilbene disulfonate binding cause anion transport inhibition, identification of the DNDS binding regions may localize the substrate binding region of the protein. Cysteine residues were introduced into 27 sites in the extracellular loop regions of an otherwise cysteineless form of AE1, called AE1C(-). The ability to label these residues with biotin maleimide [3-(N-maleimidylpropionyl)biocytin] was then measured in the absence and presence of DNDS. DNDS reduced the ability to label residues in the regions around G565, S643-M663, and S731-S742. We interpret these regions either as (i) part of the DNDS binding site or (ii) distal to the binding site but undergoing a conformational change that sequesters the region from accessibility to biotin maleimide. DNDS alters the conformation of residues outside the plane of the bilayer since the S643-M663 region was previously shown to be extramembranous. Upon binding DNDS, AE1 undergoes conformational changes that can be detected in extracellular loops at least 20 residues away from the hydrophobic core of the lipid bilayer. We conclude that the TM7-10 region of AE1 is central to the stilbene disulfonate and substrate binding region of AE1.     

Voltage-gated anion channel of the electric organ of Narke japonica incorporated into planar bilayers

Voltage-gated anion channels in vesicles prepared from the electric organ of Narke japonica were studied using two methods. Ionic permeability was measured by the light scattering method, which could be used to measure the ion permeation of whole vesicles but only at a time scale of slower than about 0.1 s. The single channel conductances and permeability ratios for various ions were measured after fusing the vesicles to phospholipid bilayers. Both sets of results coincided, indicating that the anion channels observed with the planar bilayer method are the major route for anion passage in these vesicles. The channels showed anion selectivity and did not allow the permeation of cations such as K+ and choline+. The single channel conductance was 18 pS in 0.1 M Cl-. SCN- inhibited the conductance in a voltage-dependent reversible manner on both sides of a channel. SCN- may bind to the Cl- binding site in a channel and thus block it. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocked a channel on the cis (extracellular) side irreversibly. The number of anion channels per vesicle was estimated to be about 50. It was also shown that all anion channels in the vesicles were open at the very instance of fusion with planar membranes.     

Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates

The anion transporter from human red blood cells, band 3, has been expressed in Xenopus laevis frog oocytes microinjected with mRNA prepared from the cDNA clone. About 10% of the protein is present at the plasma membrane as determined by immunoprecipitation of covalently bound 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) with anti-DIDS antibody. The expressed band 3 transport chloride at a rate comparable to that in erythrocytes. Transport of chloride is inhibited by stilbene disulfonates, niflumic acid, and dipyridamole at concentrations similar to those that inhibit transport in red blood cells: DIDS and 4,4'-dinitro-2,2'-stilbene disulfonate inhibit chloride uptake with Kiapp of 34 nM and 2.5 microM, respectively. Lysine 539 has been tentatively identified as the site of stilbene disulfonate binding. Site-directed mutagenesis of this lysine to five different amino acids has no effect on transport. Inhibition by stilbene disulfonates or their covalent binding was not affected when Lys-539 was substituted by Gln, Pro, Leu, or His. However, substitution by Ala resulted in weaker inhibition and covalent binding. These results indicate that lysine 539 is not part of the anion transport site and that it is not essential for stilbene disulfonate binding and inhibition.     

Chloride channel function is linked to epithelium-dependent airway relaxation

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl- secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl- channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl- in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl- secretion using Cl- free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl- channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl- channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl- channel other than CFTR is active in the SP response. This work introduces a possible role for Cl- pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.     

Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes. Comparison of two different affinity labels: 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid and brominated taurodehydrocholic acid

4,4'-Diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311-330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127-135). [3H]H2DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [3H]H2DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., M?ller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254-261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.