4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Cystic-Fibrosis

The basic defect in cystic fibrosis is the chloride impermeability of the plasmalemma in different cells. A candidate for the chloride channel, thought to be affected in the syndrome, is "Porin 31HL" recently described by us. The molecule is i) expressed in the plasmalemma of different cells, it has ii) a molecular mass of 31,000 Daltons, it shows iii) high conductance in artificial membranes and it can be iv) modified by 4,4'-Diisothiocyanatostilbene-2,2'-disulfonate. A porin in the outer membrane of cells should furthermore v) be regulated by modulators. All these characters of "Porin 31HL" correspond to those given in literature for chloride channels. The regulation of the channels can be explained by a two component flip flop model.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Cell Membrane Permeability; Chlorides; Cystic Fibrosis; Humans; Ion Channels; Membrane Lipids; Membrane Proteins; Molecular Weight; Porins; Voltage-Dependent Anion Channel 1; Voltage-Dependent Anion Channels

Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl(-)) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl(-) transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl(-) channels to mediate Cl(-) transport across lipid bilayer membranes is capable of restoring Cl(-) permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl(-) channel dysfunction.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cell Line; Chloride Channels; Chlorides; Cystic Fibrosis; Electrophysiology; Epithelial Cells; Humans; Lipopolysaccharides; Mice; Niflumic Acid; ortho-Aminobenzoates; Tumor Necrosis Factor-alpha

This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using 36Cl and 125I efflux. Separate Ca(2+)- and cAMP-dependent Cl- currents were identified. Ca(2+)-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. Ionomycin (2 microM) increased this current from 357 +/- 72 pA to 1,192 +/- 414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of 125I > 36Cl in 15:15 studies. Ionomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) (150 microM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 microM) or cpt-cAMP (500 microM) increased this current from 189 +/- 50 pA to 784 +/- 196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of 36Cl > 125I in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca(2+)- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFTR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bile Ducts; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Iodides; Ionomycin; Kinetics; Liver; Male; Membrane Potentials; Membrane Proteins; Rats; Rats, Sprague-Dawley; Thionucleotides

The regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator (CFTR) is phosphorylated by protein kinase A and protein kinase C (PKC) in vivo (Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992) J. Biol. Chem. 267, 12742-12752), but so far the functional effect of the PKC-dependent phosphorylation has not been clarified. We investigated the effect of PKC on the CFTR-mediated Cl- transport by treating with phorbol 12-myristate 13-acetate (PMA), the cell line C127i stably expressing CFTR wild type (C127 CFTRw/t), or CFTR bearing the most common mutation deltaF508 (C127 CFTRdF508). We show that PMA activates Cl- efflux in C127 CFTRw/t, but not in C127 CFTRdF508 and C127i. The PMA-dependent activation of CFTR is not mediated by increase of intracellular [cAMP] and is not the result of a primary activation of a K+ conductive pathway. These results strongly suggest that PKC activates directly CFTR-mediated Cl- transport.

Topics: 1-Methyl-3-isobutylxanthine; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cell Line; Chloride Channels; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Ion Channels; Kinetics; Membrane Proteins; Mice; Protein Kinase C; Protein Kinases; Tetradecanoylphorbol Acetate; Transfection

ATP increases intracellular Ca++ ([Ca++]i) by activating different P2-purinoreceptors. Because ATP increases Cl- secretion in cystic fibrosis (CF)-affected epithelia, the current study was designed to establish the link between these two events. Studies were done in epithelial, human MCF-7 breast tumor cells in which the presence of mRNA transcripts encoding CF transmembrane conductance regulator was initially established. Changes in [Ca++]i were measured in single cells by fluorescence microscopy; anion transport was measured by 125I efflux. ATP stimulated concentration-dependent increases in [Ca++]i and 125I efflux from MCF-7 cells. The relative order of agonist potency of various selective P2-purinoreceptor agonists in increasing [Ca++]i and 125I efflux was: UTP > or = ATP > ADP = AMP; 2-chloro-ATP, 2-methylthio-ATP and AMP-phencyclidine were considerably less potent than ATP. The Ca++ ionophore ionomycin increased both intracellular [Ca++]i and 125I secretion. Exposing cells to the intracellular chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA)-acetoxymethylester decreased (AM) decreased ATP- and ionomycin-stimulated 125I efflux. Extracellular EGTA did not alter the Ca++ response to ATP, but inhibited the response to ionomycin. The chelator inhibited both ATP- and ionomycin-induced 125I secretion. Exposure of cells to nifedipine did not affect the responsiveness of MCF-7 cells to ATP. The anion transport antagonist 4,4'-diisothiocyananatostilbene-2,2'-disulfonic acid partially inhibited ATP- and cationophore-stimulated increases in [Ca++]i and 125I secretion. The data suggest that activation of P2 receptors in MCF-7 cells leads to an increase in anion transport as a result of the ability of ATP to increase [Ca++]i; moreover, anion channel antagonists may produce their inhibitory effect on 125I secretion, in part, by blocking agonist-induced intracellular Ca++ signaling.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Breast Neoplasms; Calcium; Chloride Channels; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dose-Response Relationship, Drug; Humans; Iodine Radioisotopes; Ion Channels; Membrane Proteins; Receptors, Purinergic; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

We studied the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) expression in NIH-3T3 fibroblasts as a model for the effects of virally transduced CFTR expression in non-epithelial cells. Fibroblasts were infected with a retrovirus vector that contained the human CFTR and neor cDNAs. We selected and expanded G418-resistant clones that encompassed a range of CFTR expression. CFTR-mediated Cl-conductance function was measured as whole cell current, and CFTR protein was quantitated by immunoblot analysis. Overall, there was a good relationship between CFTR protein levels and CFTR-mediated Cl- conductance. Some clones had consistently high basal levels of CFTR-mediated Cl- conductance. This variation in function was partially explained by CFTR protein levels and was not due to clonal variation in cAMP metabolism. High levels of CFTR expression were associated with depolarization of fibroblast membrane potential. The CFTR-expressing clones with the largest basally active CFTR Cl- conductances and the most depolarized membrane potentials also exhibited slower growth rates. These results suggest that potential side effects of gene replacement therapy for cystic fibrosis include functional consequences of CFTR expression in non-epithelial cells.

Topics: 3T3 Cells; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Blotting, Western; Chloride Channels; Chlorides; Clone Cells; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Fibroblasts; Humans; Ion Channels; Membrane Potentials; Membrane Proteins; Mice; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Cells, Cultured; Chloride Channels; Chlorides; Cystic Fibrosis; Electric Conductivity; Epithelium; Humans; In Vitro Techniques; Ion Channel Gating; Ion Channels; Membrane Potentials; Membrane Proteins; Respiratory System

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in stage V/VI Xenopus oocytes by injection of cRNA transcribed in vitro from a pBluescript vector containing 6.2-kb wild-type cDNA. This clone was also used for the preparation of antisense RNA. Double-electrode voltage clamp was employed to measure transmembrane currents. In sense RNA-injected oocytes, cAMP depolarized the membrane potential (Vm) from -52 to -31 mV and increased membrane conductance (Gm) 10-fold. However, cAMP had no effect on Vm or Gm in uninjected oocytes or in oocytes injected with antisense RNA. The endogenous Ca-activated Cl currents of control oocytes were abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50 microM) or bath Cl replacement. In contrast, the cAMP-stimulated currents of CFTR-expressing oocytes were DIDS insensitive and were inhibited only approximately 50% when bath Cl was replaced by gluconate or glutamate. In addition, the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; 50 microM) and diphenylamine-2-carboxylic acid (DPC; 3 mM) reduced the cAMP-evoked currents by only approximately 10%. The stimulated currents of CFTR-expressing oocytes were reduced approximately 30% by 10 mM Ba, suggesting that the Cl-independent current component is due to an increase in K conductance. Our results indicate that expression of CFTR in Xenopus oocytes produces a cAMP-activated Cl current. The Cl-independent current may represent a regulatory action of CFTR on K conductance pathways or a secondary response of the oocyte membrane to the high Cl conductance induced by CFTR expression.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Alkaline Phosphatase; Animals; Calcium; Cells, Cultured; Chlorides; Cloning, Molecular; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Humans; Membrane Potentials; Membrane Proteins; Oocytes; RNA; Transcription, Genetic; Xenopus laevis

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

Topics: 3T3 Cells; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chloride Channels; CHO Cells; Cricetinae; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Epithelial Cells; Epithelium; Female; Fibroblasts; Membrane Proteins; Mice; Ovary

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; B-Lymphocytes; Bacterial Outer Membrane Proteins; Biological Transport, Active; Cell Line; Chloride Channels; Chlorides; Cystic Fibrosis; Humans; Ion Channels; Lipid Bilayers; Membrane Potentials; Membrane Proteins; Porins

Pancreatic acini of control and reserpine-treated rats were incubated with the isotopic tracer 36Cl to compare Cl accumulation in the absence and presence of secretagogues and transport inhibitors. Two phases of Cl accumulation were ascertained in resting control cells: an initial rate (0-5 min) and a steady state level (10-30 min) of accumulation. Both phases were enhanced by acetylcholine (1 microM) and caerulein (10 nM), but not by 10 nM vasointestinal peptide or 10 microM forskolin. Exposure to 1 mM DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid) inhibited both phases of Cl accumulation, whereas exposure to 1 mM amiloride had a delayed effect on the initial rate and reduced the steady state phase in both resting (unstimulated) or acetylcholine-stimulated cells. Furosemide (1 mM) had no effect on Cl accumulation when added to the cells just before tracer, but reduced it when added 10 min before. Neither the initial phase nor the steady state level of Cl accumulation were enhanced by acetylcholine in acini of reserpine-treated rats and the effect of DIDS on the initial phase was smaller than in control cells. Continued exposure to this inhibitor resulted, furthermore, in a significantly larger steady state Cl content. The inhibitory effects of amiloride and of a 10-min preincubation with furosemide were similar to those observed in control cells. These results suggest that Cl accumulates in rat pancreatic acini by way of DIDS-sensitive mechanisms that are activated by Ca2+-mediated, but not by cAMP-mediated, secretagogues. These mechanisms are altered in acini of reserpine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetylcholine; Amiloride; Animals; Biological Transport; Ceruletide; Chlorides; Colforsin; Cystic Fibrosis; Disease Models, Animal; Furosemide; Kinetics; Male; Pancreas; Rats; Rats, Inbred Strains; Reserpine; Vasoactive Intestinal Peptide

Sulfate uptake into skin fibroblasts from patients with cystic fibrosis is increased. Sulfate transport studies were carried out in skin fibroblasts isolated from age/sex matched cystic fibrosis and normal subjects. Sulfate transport occurred mainly via a carrier-mediated proton-stimulated S04(2)-/Cl-exchange. The capacity (Vmax) of the uptake system operating at physiological concentrations of sulfate was stimulated in cystic fibrosis, but the affinity of the carrier for sulfate was not altered.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Bicarbonates; Biological Transport; Cells, Cultured; Chlorides; Cystic Fibrosis; Fibroblasts; Humans; Kinetics; Reference Values; Skin; Sulfates

Chloride permeability in 9 cystic fibrosis- and 11 normal-skin fibroblast lines has been investigated. Chloride efflux, under steady-state conditions, involves two intracellular compartments characterized by slow- and fast-rate constants of efflux. We show here that the fast rate constant in cystic fibrosis cells is reduced by 25% in comparison with controls. The data presented support recent studies indicating that isolated sweat glands and respiratory epithelia of patients suffering from cystic fibrosis have an unusual low permeability to chloride ions compared to control epithelia. It is concluded that variation in chloride transport can successfully be studied in cultured fibroblasts, which are not directly involved in the pathology of the disease.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Cell Membrane Permeability; Chlorides; Cold Temperature; Cystic Fibrosis; Fibroblasts; Humans; Kinetics; Mathematics; Skin

The chloride permeability of airway and sweat ductal epithelium of cystic fibrosis (CF) patients is decreased. This abnormality could represent an intrinsic characteristic of the epithelial cell or the response to a tonic extrinsic stimulus, in vivo. We cultured airway epithelial cells derived from CF and non-CF individuals under identical conditions that were free from donor-specific factors. Differences in the characteristics of cells that multiplied under these circumstances are unlikely to reflect the effects of extrinsic modulation present in the host. After 8-12 days in culture, the cells of CF and non-CF patients were similar in morphology and intracellular electrolyte content, but the CF cultures took up chloride at a reduced rate. The difference could not be attributed to a higher intracellular potential in CF cells or to the presence of a stilbene anion-sensitive chloride-chloride exchange in non-CF cells. We conclude that epithelial cells from CF patients grown in the absence of extracellular factors of the host express reduced cellular chloride permeability, a defect similar to that found in vivo and in freshly excised nasal epithelium.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Biological Transport; Cell Membrane Permeability; Cells, Cultured; Chlorides; Cystic Fibrosis; Epithelium; Humans; Intracellular Fluid; Nasal Mucosa; Onium Compounds; Potassium; Sodium; Trityl Compounds