stilbenes and 4-4--dinitro-2-2--stilbenedisulfonic-acid

Chloride channels are ubiquitously distributed, biophysically varied and functionally diverse. Despite the known contribution of chloride channels to the physiology of various cell types and the pathology of several diseases, high affinity ligands are not available to study these channels. Here we report the iterative and integrated use of ion channel kinetic analysis and computational chemical methods in the development of high affinity blockers of the outwardly rectifying chloride channel (ORCC). Kinetic analysis, with emphasis on estimation of the block time constant as determined from critical closed time plots, was used to guide the synthesis of new disulfonic stilbene derivatives. Computational chemical methods were used to deduce the important features of the disulfonic stilbene molecule necessary for potent blockade of ORCC and ultimately led to the discovery of the calixarenes. Para-sulfonated calixarenes were found to be potent blockers of ORCC with subnanomolar inhibition constants and exceptionally long block times.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Chloride Channels; Electrochemistry; Humans; Kinetics; Models, Molecular; Stilbenes

The hepatocyte growth factor receptor (HGFR or c-Met) is a driver of multiple cancer subtypes. While there are several c-Met inhibitors in development, few have been approved for clinical use, warranting the need for continued research and development of c-Met targeting therapeutic modalities. The research presented here demonstrates a particular class of compounds known as isothiocyanatostilbenes can act as c-Met inhibitors in multiple cancer cell lines. Specifically, we found that 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) had c-Met inhibitory effective doses in the low micromolar range while 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS) exhibited IC50s 100 to 1000 fold higher. These compounds displayed much greater selectivity for inhibiting c-Met activation compared to similar receptor tyrosine kinases. In addition, DIDS and H2DIDS reduced hepatocyte growth factor (HGF)-induced, but not epidermal growth factor (EGF)-induced, cell scattering, wound healing, and 3-dimensional (3D) proliferation of tumor cell spheroids. In-cell and cell-free assays suggested that DIDS and H2DIDS can inhibit and reverse c-Met phosphorylation, similar to SU11274. Additional data demonstrated that DIDS is tolerable in vivo. These data provide preliminary support for future studies examining DIDS, H2DIDS, and derivatives as potential c-Met therapeutics.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Female; Hepatocyte Growth Factor; Humans; Mice, Nude; Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-met; Stilbenes; Time Factors; Xenograft Model Antitumor Assays

The inferior olivary nucleus (IO) forms the gateway to the cerebellar cortex and receives feedback information from the cerebellar nuclei (CN), thereby occupying a central position in the olivo-cerebellar loop. Here, we investigated the feedback input from the CN to the IO in vivo in mice using the whole-cell patch-clamp technique. This approach allows us to study how the CN-feedback input is integrated with the activity of olivary neurons, while the olivo-cerebellar system and its connections are intact. Our results show how IO neurons respond to CN stimulation sequentially with: i) a short depolarization (EPSP), ii) a hyperpolarization (IPSP) and iii) a rebound depolarization. The latter two phenomena can also be evoked without the EPSPs. The IPSP is sensitive to a GABA(A) receptor blocker. The IPSP suppresses suprathreshold and subthreshold activity and is generated mainly by activation of the GABA(A) receptors. The rebound depolarization re-initiates and temporarily phase locks the subthreshold oscillations. Lack of electrotonical coupling does not affect the IPSP of individual olivary neurons, nor the sensitivity of its GABA(A) receptors to blockers. The GABAergic feedback input from the CN does not only temporarily block the transmission of signals through the IO, it also isolates neurons from the network by shunting the junction current and re-initiates the temporal pattern after a fixed time point. These data suggest that the IO not only functions as a cerebellar controlled gating device, but also operates as a pattern generator for controlling motor timing and/or learning.

Topics: Action Potentials; Animals; Cerebellar Nuclei; Feedback, Physiological; GABA-A Receptor Antagonists; Learning; Mice; Mice, Inbred C57BL; Microelectrodes; Neural Pathways; Neurons; Olivary Nucleus; Patch-Clamp Techniques; Receptors, GABA-A; Stilbenes; Synaptic Transmission

Despite the importance of corticocortical connections, few published studies have investigated the functional, synaptic properties of such connections in any species, because most studies have been purely anatomical or aimed at functional features other than synaptic properties. We recently published a study of synaptic properties of connections between the primary and secondary cortical auditory areas in brain slices from the mouse, and, in the present study, we aimed to extend this by performing analogous studies of the primary and secondary visual areas (V1 and V2). We found effectively the same results. That is, connections between V1 and V2 in both directions were quite similar; in each case, the glutamatergic inputs could be classified as one of two types, Class 1B (formerly "driver") and Class 2 (formerly "modulator"). There is a clear laminar correlation for these different inputs, in terms of both the laminae of origin and those in which the recorded cells were located. Our data suggest a common pattern to the functional organization of corticocortical connectivity in the mouse cortex.

Topics: Animals; Animals, Newborn; Biophysics; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA Antagonists; Glutamic Acid; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Male; Mice; Mice, Inbred BALB C; Nerve Net; Neurons; Patch-Clamp Techniques; Photic Stimulation; Receptors, Metabotropic Glutamate; Stilbenes; Synapses; Visual Cortex; Visual Pathways

Chronic stress is a major health concern, often leading to depression, anxiety, or when severe enough, posttraumatic stress disorder. While many studies demonstrate that the amygdala is hyperresponsive in patients with these disorders, the cellular neurophysiological effects of chronic stress on the systems that underlie psychiatric disorders, such as the amygdala, are relatively unknown.. In this study, we examined the effects of chronic stress on the activity and excitability of amygdala neurons in vivo in rats. We used in vivo intracellular recordings from single neurons of the lateral amygdala (LAT) to measure neuronal properties and determine the cellular mechanism for the effects of chronic stress on LAT neurons.. We found a mechanism for the effects of chronic stress on amygdala activity, specifically that chronic stress increased excitability of LAT pyramidal neurons recorded in vivo. This hyperexcitability was caused by a reduction of a regulatory influence during action potential firing, facilitating LAT neuronal activity. The effects of stress on excitability were occluded by agents that block calcium-activated potassium channels and reversed by pharmacological enhancement of calcium-activated potassium channels.. These data demonstrate a specific channelopathy that occurs in the amygdala after chronic stress. This enhanced excitability of amygdala neurons after chronic stress may explain the observed hyperresponsiveness of the amygdala in patients with posttraumatic stress disorder and may facilitate the emergence of depression or anxiety in other patients.

Topics: Action Potentials; Adrenal Glands; Amygdala; Animals; Calcium Channel Agonists; Calcium Channel Blockers; Chronic Disease; Male; Maze Learning; Neurons; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Stilbenes; Stress, Psychological

This study uses 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNS) wastewater to produce paramycin (4-amino-2-hydroxybenzoic acid), an antitubercular agent and important pharmaceutical intermediate. The high concentrations of aromatic sulfonic acids contained in the wastewater, derived from a DNS production facility, have been transformed to paramycin in yields of more than 85%. This waste-disposal strategy, which combines oxidation using NaClO, reduction using iron powder, and subsequent alkaline fusion with NaOH, has been proven to be successful in dealing with ton-scale DNS wastewater. Compared with common treatment methods, which usually involve degrading the compounds, this new method recycles most of the aromatic sulfonic acids in the wastewater to produce paramycin. This effectively solves the associated environment problems associated with DNS wastewater and is also potentially profitable. The present approach could also lead to alternative solutions for dealing with other industrial wastewaters generated from oxidative coupling reactions of nitro-substituted toluenes to the corresponding substituted stilbenes.

Topics: Aminosalicylic Acid; Antitubercular Agents; Oxidation-Reduction; Stilbenes; Waste Disposal, Fluid; Water Pollutants, Chemical

The pharmacology of Caenorhabditis elegans glutamate-gated chloride (GluCl) channels was determined by making intracellular voltage-clamp recordings from Xenopus oocytes expressing GluCl subunits. As previously reported (Cully et al. 1994), GluClalpha1beta responded to glutamate (in a picrotoxin sensitive manner) and ivermectin, while GluClbeta responded only to glutamate and GluClalpha1 only to ivermectin. This assay was used to further investigate the action of chloride channel compounds. The arylaminobenzoate, NPPB, reduced the action of glutamate on the heteromeric GluClalpha1beta channel (IC(50) 6.03 +/- 0.81 microM). The disulphonate stilbene, DNDS, blocked the effect of both glutamate and ivermectin on GluClalpha1beta channels, the action of glutamate on GluClbeta subunits, and the effect of ivermectin on GluClalpha1 subunits (IC(50)s 1.58-3.83 microM). Surprisingly, amobarbital and pentobarbital, otherwise known as positive allosteric modulators of ligand-gated chloride channels, acted as antagonists. Both compounds reduced the action of glutamate on the GluClalpha1beta heteromer (IC(50)s of 2.04 +/- 0.5 and 17.56 +/- 2.16 microM, respectively). Pentobarbital reduced the action of glutamate on the GluClbeta homomeric subunit with an IC(50) of 0.59 +/- 0.09 microM, while reducing the responses to ivermectin on both GluClalpha1beta and GluClalpha1 with IC(50)s of 8.7 +/- 0.5 and 12.9 +/- 2.5 microM, respectively. For all the antagonists, the mechanism is apparently non-competitive. The benzodiazepine, flurazepam had no apparent effect on these glutamate- and ivermectin-gated chloride channel subunits. Thus, arylaminobenzoates, disulphonate stilbenes, and barbiturates are non-competitive antagonists of C. elegans GluCl channels.

Topics: Animals; Anthelmintics; Barbiturates; Benzodiazepines; Caenorhabditis elegans; Chloride Channels; Female; Glutamic Acid; Ivermectin; Nitrobenzoates; Oocytes; Patch-Clamp Techniques; Picrotoxin; Stilbenes; Xenopus

Plasma membrane potentials gate the ion channel conductance that controls external signal-induced neuronal functions. We found that diffusible guidance molecules caused membrane potential shifts that resulted in repulsion or attraction of Xenopus laevis spinal neuron growth cones. The repellents Sema3A and Slit2 caused hyperpolarization, and the attractants netrin-1 and BDNF caused depolarization. Clamping the growth-cone potential at the resting state prevented Sema3A-induced repulsion; depolarizing potentials converted the repulsion to attraction, whereas hyperpolarizing potentials had no effect. Sema3A increased the intracellular concentration of guanosine 3',5'-cyclic monophosphate ([cGMP]i) by soluble guanylyl cyclase, resulting in fast onset and long-lasting hyperpolarization. Pharmacological increase of [cGMP](i) caused protein kinase G (PKG)-mediated depolarization, switching Sema3A-induced repulsion to attraction. This bimodal switch required activation of either Cl(-) or Na+ channels, which, in turn, regulated the differential intracellular Ca2+ concentration increase across the growth cone. Thus, the polarity of growth-cone potential shifts imposes either attraction or repulsion, and Sema3A achieves this through cGMP signaling.

Topics: Animals; Brain-Derived Neurotrophic Factor; Calcium; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Growth Cones; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Membrane Potentials; Microinjections; Nerve Growth Factors; Nerve Tissue Proteins; Netrin-1; Neurons; Patch-Clamp Techniques; Potassium; Semaphorin-3A; Signal Transduction; Stilbenes; Time Factors; Tumor Suppressor Proteins; Xenopus laevis

Ischemia-induced extracellular glutamate accumulation and the subsequent excitotoxicity contribute significantly to ischemic brain injury. Volatile anesthetics have been shown to reduce ischemic brain injury. Here, we showed that oxygen-glucose deprivation (OGD, to simulate ischemia in vitro) increased extracellular glutamate accumulation in the corticostriatal slices of adult rats. This increased accumulation was reduced by dihydrokinate, a glutamate transporter type 2 inhibitor, and 4,4'-dinitrostilbene-2,2'-disulfonic acid, a blocker for volume-activated anion channels. The volatile anesthetics isoflurane, sevoflurane and desflurane at clinically relevant concentrations did not affect the OGD-induced extracellular glutamate accumulation from brain slices of adult rats. Isoflurane also did not change the OGD-induced extracellular glutamate accumulation from brain slices of newborn/young rats. These results suggest that the OGD-induced glutamate accumulation involves reversed transport of glutamate via glutamate transporters and volume-activated anion channels. Volatile anesthetics may not inhibit this extracellular glutamate accumulation.

Topics: Amino Acid Transport System X-AG; Anesthetics, Inhalation; Animals; Animals, Newborn; Anions; Brain; Cell Hypoxia; Chromatography, High Pressure Liquid; Desflurane; Enzyme Inhibitors; Excitatory Amino Acid Transporter 2; Extracellular Fluid; Glucose; Glutamic Acid; In Vitro Techniques; Ion Channels; Isoflurane; Male; Methyl Ethers; Neurons; Oxygen; Rats; Rats, Sprague-Dawley; Sevoflurane; Stilbenes

Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current (I(sc); 171 +/- 21 microA/cm(2)) and lumen-negative potential difference (-10.1 +/- 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to I(sc). In response to 2 microM forskolin, I(sc) exceeded 270 microA/cm(2), and impedance estimates of the apical membrane resistance decreased from 200 Omega.cm(2) to 13 Omega.cm(2). The forskolin-induced I(sc) was dependent on extracellular HCO(3)(-) and was blocked by serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl(-) ion substitution had little effect. Serosal trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba(2+) reduced I(sc), consistent with the inhibition of cAMP-dependent K(+) channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO(3)(-) entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K(+) channel activation.

Topics: Acetazolamide; Adenylyl Cyclases; Animals; Bicarbonates; Carbonic Anhydrase Inhibitors; Chlorides; Colforsin; Cyclic AMP; Cyclooxygenase Inhibitors; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Impedance; Enzyme Activators; Gallbladder; Indomethacin; Male; Membrane Potentials; Potassium; Potassium Channels; Sciuridae; Sodium; Sodium-Bicarbonate Symporters; Stilbenes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40+/-0.070min(-1) and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using (1)H NMR, had a K(m) of 1.49+/-0.16mM and V(max) of 2.61+/-0.08micromolL(-1)min(-1). Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of approximately 10microM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were approximately 5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetates; Acetylation; Acetylcysteine; Cystine; Erythrocytes; Glutathione; Humans; Kinetics; Stilbenes; Substrate Specificity

Neuronal death associated with cerebral ischemia and hypoglycemia is related to increased release of excitatory amino acids (EAA) and energy failure. The intrahippocampal administration of the glycolysis inhibitor, iodoacetate (IOA), induces the accumulation of EAA and neuronal death. We have investigated by microdialysis the role of exocytosis, glutamate transporters and volume-sensitive organic anion channel (VSOAC) on IOA-induced EAA release. Results show that the early component of EAA release is inhibited by riluzole, a voltage-dependent sodium channel blocker, and by the VSOAC blocker, tamoxifen, while the early and late components are blocked by the glutamate transport inhibitors, L-trans-pyrrolidine 2,4-dicarboxylate (PDC) and DL-threo-beta-benzyloxyaspartate (DL-TBOA); and by the VSOAC blocker 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Riluzole, DL-TBOA and tamoxifen did not prevent IOA-induced neuronal death, while PDC and DNDS did. The VSOAC blockers 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) and phloretin had no effect either on EAA efflux or neuronal damage. Results suggest that acute inhibition of glycolytic metabolism promotes the accumulation of EAA by exocytosis, impairment or reverse action of glutamate transporters and activation of a DNDS-sensitive mechanism. The latest is substantially involved in the triggering of neuronal death. To our knowledge, this is the first study to show protection of neuronal death by DNDS in an in vivo model of neuronal damage, associated with deficient energy metabolism and EAA release, two conditions involved in some pathological states such as ischemia and hypoglycemia.

Topics: Animals; Aspartic Acid; Brain Ischemia; Cell Death; Energy Metabolism; Excitatory Amino Acids; Exocytosis; Extracellular Fluid; Glycolysis; Hippocampus; Male; Microdialysis; Nerve Degeneration; Nitrobenzoates; Phloretin; Rats; Rats, Wistar; Riluzole; Stilbenes; Tamoxifen; Vesicular Glutamate Transport Proteins; Voltage-Dependent Anion Channels

Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antiporters; Bicarbonates; Blotting, Northern; Blotting, Western; Carbachol; Carbonic Anhydrase Inhibitors; Cell Line; Chloride Channels; Colon; Cyclic AMP; Epithelial Cells; Humans; Immunohistochemistry; Membrane Transport Proteins; Muscarinic Agonists; Quaternary Ammonium Compounds; Rats; Reverse Transcriptase Polymerase Chain Reaction; Stilbenes; Sulfate Transporters

We evaluated the relationship between apical surface fluid (ASF) and protein secretion in Calu-3 cells grown at an air-liquid interface. Calu-3 monolayers responded to forskolin, a cystic fibrosis transmembrane regulator (CFTR) channel agonist, by secreting a significant amount of ASF. Such a response from Calu-3 monolayers was not observed with CFTR channel blockers glybenclamide and DPC. Other ion channel mediators, PGF-2alpha, PMA, DNDS, and DIDS, had no effect on Calu-3 ASF secretion. Forskolin decreased Calu-3 protein secretion and glybenclamide increased protein secretion. Similarly, forskolin decreased Calu-3 lysozyme secretion, whereas glybenclamide and DPC increased lysozyme secretion. We observed significant changes in Calu-3 fluid and protein secretions with ion channel mediators known to alter CFTR activity. Our results demonstrate a functional link between fluid and protein secretions in Calu-3 apical surface and suggested a possible involvement of CFTR in these processes.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anti-Arrhythmia Agents; Body Fluids; Cell Line; Cell Polarity; Colforsin; Cystic Fibrosis Transmembrane Conductance Regulator; Dinoprost; Epithelial Cells; Glyburide; Humans; Muramidase; Oxytocics; Respiratory Mucosa; Stilbenes; Tetradecanoylphorbol Acetate

The human NBC1 (SLC4A4) gene encodes the electrogenic sodium bicarbonate cotransporters kNBC1 and pNBC1, which are highly expressed in the kidney and pancreas, respectively. The HCO3-:Na+ stoichiometry of these cotransporters is an important determinant of the direction of ion flux. Recently we showed in a mouse proximal tubule (mPCT) cell line expressing kNBC1, that 8-Br-cAMP shifts the stoichiometry of the cotransporter from 3:1 to 2:1 via protein kinase A (PKA)-dependent phosphorylation of Ser982. pNBC1 has the identical carboxy-terminal consensus phosphorylation PKA site (KKGS1026), and an additional site in its amino-terminus (KRKT49). In this study we determined the potential role of these sites in regulating the function of pNBC1. The results demonstrated that in mPCT cells expressing pNBC1, PKA-dependent phosphorylation of Ser1026 following 8-Br-cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser1026 with Asp resulted in a similar stoichiometry shift. In addition to shifting the stoichiometry, 8-Br-cAMP caused a significant increase in the 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive basolateral membrane conductance (GDS) of cells expressing pNBC1, but not kNBC1. Although, the effect did not involve phosphorylation of Thr49, which was endogenously phosphorylated, replacing this residue with Asp or Ala abolished the 8-Br-cAMP-induced increase in GDS. In the mPEC pancreatic duct cell line, where endogenous pNBC1 functions with a HCO3-:Na+ stoichiometry of 2:1, 8-Br-cAMP increased GDS by ~90 % without altering the stoichiometry or inducing phosphorylation of the cotransporter. The results demonstrate that phosphorylation of Ser1026 mediates the cAMP-dependent shift in the stoichiometry of pNBC1, whereas Thr49 plays an essential role in the cAMP-induced increase in GDS.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Algorithms; Amino Acid Substitution; Amino Acids; Animals; Bicarbonates; Carboxylic Acids; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Humans; Kinetics; Membrane Potentials; Mice; Mutagenesis; Pancreas; Phosphorylation; Sodium; Sodium-Bicarbonate Symporters; Stilbenes

Stimulation of Calu-3 epithelia with 7,8-benzoquinoline, under short circuit current conditions, produced a current increase that was completely accounted for by the net flux of chloride, measured simultaneously with 36Cl-. Nevertheless the current stimulated by 7,8-benzoquinoline was sensitive to acetazolamide, which caused up to 50 % inhibition of the stimulated current, the remainder being sensitive to the Na+-K+-2Cl- cotransport inhibitor bumetanide. The effects of acetazolamide could be mimicked by either amiloride or by the di-sodium salt of 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) added to the basolateral side of the epithelium, but their actions were not additive. Amiloride was needed in sufficient concentration to inhibit the sodium-proton exchanger NHE1. DNDS blocks both the chloride-bicarbonate exchanger AE2 and the sodium-bicarbonate transporter NBC1. However, since 7,8-benzoquinoline activates basolateral K+ channels, causing hyperpolarisation, it is unlikely NBC1 is active after addition of 7,8-benzoquinoline. The effect of DNDS is, therefore, mainly on AE2. It is concluded that chloride enters the basolateral aspect of the cells using the Na+-K+-2Cl- cotransporter and a parallel arrangement of NHE1 with AE2, these latter two being sensitive to acetazolamide because of their association with the cytoplasmic form of carbonic anhydrase CAII. The effects of acetazolamide could be mimicked by removal of HCO3-/CO2 from the bathing medium, and furthermore showed that the NHE1-AE2 mechanism is particularly important when the transport rate is high. Thus part of the current stimulated by 7,8-benzoquinoline and inhibited by acetazolamide or HCO3-/CO2 removal can be said to represent bicarbonate-dependent chloride secretion.

Topics: Acetazolamide; Amiloride; Anions; Bicarbonates; Bumetanide; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Cell Line, Tumor; Cell Membrane; Chlorides; Epithelium; Humans; Hydrogen-Ion Concentration; Intracellular Membranes; Lung; Quinolines; Stilbenes

We have shown that diethyl pyrocarbonate (DEPC) inhibits band 3-mediated anion exchange and that the inhibition occurs only when histidine residue(s) is (are) modified with DEPC from the cytosolic surface of resealed ghosts [Izuhara et al. (1989) Biochemistry 28, 4725-4728]. In the present study, we have identified the DEPC-modified histidine residue as His834 using liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). This mild, rapid, sensitive, and quantitative method was successfully applied to analysis of the unstable DEPC-histidine adduct. The DEPC modification of His834 was pH dependent and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) sensitive as previously shown. After DEPC modification, band 3-mediated anion exchange is inhibited. Consistent with previous results, we confirmed that His834 was located on the cytosolic side of the membrane and the DEPC modification of His834 had allosteric effects on the extracellular DNDS-binding site of band 3. Therefore, we conclude that His834 is located at the cytosolic surface of band 3 and is an essential residue for band 3-mediated anion exchange. We will discuss important roles of the region from TM12 to TM14 in the conformational changes that occur during the band 3-mediated anion exchange.

Topics: Anion Exchange Protein 1, Erythrocyte; Anions; Chromatography, High Pressure Liquid; Cytosol; Diethyl Pyrocarbonate; Erythrocyte Membrane; Histidine; Humans; Ion Transport; Peptide Fragments; Peptide Mapping; Protein Conformation; Spectrometry, Mass, Electrospray Ionization; Stilbenes; Structure-Activity Relationship; Trypsin

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.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anions; Chlorides; Cystic Fibrosis Transmembrane Conductance Regulator; Dose-Response Relationship, Drug; Electric Conductivity; Glyburide; Humans; In Vitro Techniques; Male; Membrane Potentials; Nitrobenzoates; ortho-Aminobenzoates; Peptide Fragments; Phosphorylation; Reproducibility of Results; Sensitivity and Specificity; Stilbenes; Sweat Glands

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na(+), HCO, and Cl(-) for a full response. HCO-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na(+)-driven, HCO-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 base equivalents per Na(+). Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338-391 and 928-1035) identified a single band of ~145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Base Sequence; Bicarbonates; Carbonic Anhydrase Inhibitors; Cells, Cultured; Chlorides; Colforsin; Cyclic AMP; Diffusion Chambers, Culture; Epithelial Cells; Epitopes; Immunoblotting; Ion Transport; Male; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Sodium; Sodium-Bicarbonate Symporters; Stilbenes; Swine; Vas Deferens

The Calu-3 human cell line exhibits features of submucosal gland serous cells and secretes HCO(3)(-). The aim of this study was to identify the HCO(3)(-) transporters present in these cells by studying their role in the regulation of intracellular pH (pH(i)). Calu-3 cells were grown on coverslips, loaded with the pH-sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pH(i). Cells were acidified with NH(4)Cl (25 mM, 1 min) and pH(i) recovery recorded. In the absence of HCO(3)(-), initial recovery was 0.208 +/- 0.016 pH units min(-1) (n = 37). This was almost abolished by removal of extracellular Na(+) and by amiloride (1 mM), consistent with the activity of a Na(+)-H(+) exchanger (NHE). In the presence of HCO(3)(-) and CO(2), recovery (0.156 +/- 0.018 pH units min(-1)) was abolished (reduced by 91.8 +/- 6.7 %, n = 7) by removal of Na(+) but only attenuated (by 63.3 +/- 5.8 %, n = 9) by amiloride. 4,4-Dinitrostilbene-2,2-disulfonic acid (DNDS) inhibited recovery by 45.8 +/- 5.0 % (n = 7). The amiloride-insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K(+)] in the presence of either valinomycin or the K(+) channel opener 1-EBIO. In addition, forskolin (10 microM), which activates the cystic fibrosis transmembrane conductance regulator Cl(-) conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl(-) trebled pH(i) recovery rates, suggesting that an electroneutral, DNDS-sensitive, Cl(-)-HCO(3)(-) exchanger together with a NHE may be involved in pH(i) regulation and HCO(3)(-) secretion in these cells. RT-PCR detected the expression of the electrogenic Na(+)-HCO(3)(-) cotransporter NBC1 and the Cl(-)-HCO(3)(-) exchanger (AE2) but not the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1.

Topics: Amiloride; Bicarbonates; Buffers; Carbon Dioxide; Cell Line; Cell Membrane; Colforsin; HEPES; Homeostasis; Humans; Hydrogen; Hydrogen-Ion Concentration; Intracellular Membranes; Membrane Potentials; Respiratory System; Serous Membrane; Sodium; Sodium-Hydrogen Exchangers; Solutions; Stilbenes

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (I(sc)) and transepithelial resistances were 7.9 +/- 0.5 microA/cm(2) and 1,018 +/- 73 Omega.cm(2), respectively (means +/- SE; n = 12). Apical amiloride (10 microM) inhibited basal I(sc) by approximately 50%. Subsequent addition of forskolin (10 microM) increased I(sc) from 3.9 +/- 0.63 microA/cm(2) to 7.51 +/- 0.2 microA/cm(2) (n = 12). Basolateral bumetanide (100 microM) decreased forskolin-stimulated I(sc) from 7.51 +/- 0.2 microA/cm(2) to 5.62 +/- 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO secretion, blocked the remaining I(sc). Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl(-)- or HCO(-)(3)-free solutions; however, no response was seen using HCO(-)(3)- and Cl(-)-free solutions. The forskolin-stimulated I(sc) was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I(sc) across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl(-) and HCO(-)(3) secretion across rat FDLE cells mediated via CFTR.

Topics: Amiloride; Animals; Bicarbonates; Bumetanide; Cell Polarity; Cells, Cultured; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Diuretics; Epithelial Cells; Extravascular Lung Water; Immunohistochemistry; Ionophores; Membrane Potentials; Nystatin; Pulmonary Alveoli; Rats; Respiratory Mucosa; Stilbenes

Intracellular recordings were made from narrow-field, bistratified AII amacrine cells in the isolated, superfused retina-eyecup of the rabbit. Pharmacological agents were applied to neurons to dissect the synaptic pathways subserving AII cells so as to determine the circuitry generating their off-surround responses. Application of the GABA antagonists, picrotoxin, bicuculline and 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid (TPMPA) all increased the on-centre responses of AII amacrine cells, but attenuated the off-surround activity. At equal concentrations, picrotoxin was approximately twice as effective as bicuculline or TPMPA in modifying the response activity of AII amacrine cells. These results indicate that the mechanism underlying surround inhibition of AII amacrine cells includes activation of both GABA(A) and GABA(C) receptors in an approximately equal ratio. Application of the GABA antagonists also increased the size of on-centre receptive fields of AII amacrine cells. Again, picrotoxin was most effective, producing, on average, a 54 % increase in the size of the receptive field, whereas bicuculline and TPMPA produced comparable 34 and 33 % increases, respectfully. Application of the voltage-gated sodium channel blocker TTX produced effects on AII amacrine cells qualitatively similar to those of the GABA blockers. Intracellular application of the chloride channel blocker 4,4'-dinitro-stilbene-2,2'-disulphonic acid (DNDS) abolished the direct effects of GABA on AII amacrine cells. Moreover, DNDS increased the amplitude of both the on-centre and off-surround responses. The failure of DNDS to block the off-surround activity indicates that it is not mediated by direct GABAergic inhibition. Taken together, our results suggest that surround receptive fields of AII amacrine cells are generated indirectly by the GABAergic, reciprocal feedback synapses from S1/S2 amacrine cells to the axon terminals of rod bipolar cells.

Topics: Animals; Electrophysiology; Feedback; GABA Antagonists; GABA-A Receptor Antagonists; In Vitro Techniques; Nerve Net; Photic Stimulation; Presynaptic Terminals; Rabbits; Retina; Stilbenes; Visual Fields

We examined the effects of 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), an inhibitor of the chloride-bicarbonate exchangers and chloride channels, on death in cultured cerebellar granule neurons. Various stimuli, such as reduction of extracellular K+ concentration, removal of growth factors, and staurosporine treatment, induced cell death. This death was blocked by DIDS in a dose dependent manner. In the presence of DIDS, the cells exposed to such stimuli did not show DNA fragmentation, but retained the ability to exclude trypan blue and to metabolize MTT to formazan. On the other hand, pretreatment of the cells with DIDS did not show any protective effects. The neuroprotective effect of DIDS was not influenced by extracellular Na+, Cl-, HCO3- or Ca2+ concentrations, although reduction of extracellular Cl- or Ca2+ concentrations per se induced neuronal death. Other chloride-bicarbonate exchange blockers like 4-acetamido-4'-isothiocyanatostilmene-2,2'-disulfonic acid (SITS) or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) showed no significant effects on neuronal survival under these death-inducing stimuli. Dimethylamiloride, an inhibitor of the Na+/H+ exchanger, did not influence neuronal death induced by these stimuli. Cells undergoing death showed gradual intracellular acidification, and DIDS did not inhibit this response, although DIDS (2 mM) per se induced transitory acidification followed by recovery within 10 min. DIDS did not influence intracellular Ca2+ or Cl-levels during the lethal process. DIDS suppressed the cleavage of caspase-3 in the cells exposed to the death-inducing stimuli. These findings suggest that the neuroprotective effect of DIDS is mediated by a novel mechanism other than by nonselective inhibition of transporters or channels, and that DIDS blocks the death program upstream of caspases and downstream of all of the activation processes triggered by various stimuli.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Animals, Newborn; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cerebellum; Chloride Channels; Cytoprotection; Dose-Response Relationship, Drug; Drug Combinations; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Stilbenes

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.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Bronchoconstriction; Calcium Channel Blockers; Chloride Channels; Dinoprostone; Dose-Response Relationship, Drug; Gluconates; In Vitro Techniques; Isometric Contraction; Mice; Mice, Inbred CFTR; Muscle Relaxation; Nitrobenzoates; ortho-Aminobenzoates; Perfusion; Reproducibility of Results; Respiratory Mucosa; Stilbenes; Substance P; Trachea

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

Topics: Animals; Carrier Proteins; Cell Line; Cell Membrane; Electric Conductivity; Electrophysiology; Mathematics; Mice; Pancreatic Ducts; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium-Bicarbonate Symporters; Stilbenes

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.

Topics: Cell Line, Transformed; Chloride Channels; Chlorzoxazone; Cystic Fibrosis; Drug Synergism; Electric Conductivity; Glutathione; Glyburide; Humans; Intercellular Signaling Peptides and Proteins; Peptides; Potassium Channels; Stilbenes; Trachea

ATP-dependent (45)Ca uptake in rat brain microsomes was measured in intracellular-like media containing different concentrations of PO(4) and oxalate. In the absence of divalent anions, there was a transient (45)Ca accumulation, lasting only a few minutes. Addition of PO(4) did not change the initial accumulation but added a second stage that increased with PO(4) concentration. Accumulation during the second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 microM), 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS; 250 microM), and DIDS (3-5 microM); accumulation during the initial stage was unaffected. Higher concentrations of DIDS (100 microM), however, inhibited the initial stage as well. Uptake was unaffected by 20 mM Na, an activator, or 1 mM arsenate, an inhibitor of Na-PO(4) cotransport. An oxalate-supported (45)Ca uptake was larger, less sensitive to DIDS, and enhanced by the catalytic subunit of protein kinase A (40 U/ml). Combinations of PO(4) and oxalate had activating and inhibitory effects that could be explained by PO(4) inhibition of an oxalate-dependent pathway, but not vice versa. These results support the existence of separate transport pathways for oxalate and PO(4) in brain endoplasmic reticulum.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Arsenates; Brain; Calcium; Endoplasmic Reticulum; Kinetics; Male; Microsomes; Models, Biological; Niflumic Acid; Oxalates; Phosphates; Rats; Rats, Sprague-Dawley; Stilbenes

We studied the role of CFTR in the Na(+)-K(+)-pump activity of Calu-3 human airway cells. To estimate the Na(+)-K(+)-pump activity on the basolateral membrane, the ouabain-sensitive component of the short-circuit current (Isc) was measured after permeabilization of the apical membrane with nystatin, a Na(+) ionophore. The Na(+)-K(+)-pump activity was diminished by a selective CFTR blocker (glybenclamide) or nonspecific Cl(-) channel inhibitors (NPPB and DPC) but not by outwardly rectifying Cl(-) channel blockers (DNDS, DIDS). Augmentation of anion conductance by 8-bromo-cyclic AMP (8Br-cAMP, 1 mM) potentiated the Na(+)-K(+)-pump activity that was reduced by blocking CFTR or by the replacement of Cl(-) with gluconate, a less membrane-permeant anion. The Na(+)-K(+)-pump activity was unaffected by the replacement of Cl(-) with NO(-)(3) that has equal permeability through the CFTR. These results suggest that the anion movement through the CFTR may contribute to the Na(+)-K(+)-pump activity in Calu-3 cells by regulating the rate of Na(+) entry.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 8-Bromo Cyclic Adenosine Monophosphate; Anions; Bronchi; Calcium Channel Blockers; Cell Line; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Glyburide; Humans; Ionophores; Nitrobenzoates; Nystatin; ortho-Aminobenzoates; Ouabain; Sodium; Sodium-Potassium-Exchanging ATPase; Stilbenes; Time Factors

Acetylcholine (ACh) is the dominant transmitter released from inner ear efferent neurons. In frog vestibular organs, these efferent neurons synapse exclusively with type II hair cells. Hair cells isolated from the frog saccule hyperpolarize following the application of 50 microM ACh, thereby demonstrating the presence of an ACh receptor. A role for Cl(-) in the response of hair cell-bearing organs to efferent nerve activation or ACh application was suggested some years ago. Perfusion with solutions in which most of the Cl(-) was replaced by large impermeant anions decreased the cholinergic inhibition of afferent firing in the cat and turtle cochleas, and frog semicircular canal. Our previous work in the intact organ demonstrated that substitution of large impermeant anions for Cl(-) or use of Cl(-) channel blockers reduced the effect of ACh on saccular afferent firing. Using the perforated-patch clamping technique, replacement of Cl(-) by methanesulfonate, iodide, nitrate, or thiocyanate attenuated the hyperpolarizing response to ACh in hair cells isolated from the frog saccule. The chloride channel blockers picrotoxin and 4,4'-dinitrostilbene-2,2'-disulfonic acid were also tested and found to inhibit the ACh response. Thus, the present work demonstrates that the effects of Cl(-) substitutions or Cl(-) channel blockers on the ACh response in the intact saccule can be explained completely by effects on the hair cell. Evidence is also presented for the presence of the messenger RNA for a calcium-dependent chloride channel in all hair cells but especially saccular hair cells. This channel may be involved in the response to ACh. The precise role for chloride in this response, whether as a distinct ion current, as a transported ion, or as a permissive ion for other components, is discussed.

Topics: Acetylcholine; Animals; Cats; Chloride Channels; Chlorides; Hair Cells, Auditory; In Vitro Techniques; Membrane Potentials; Patch-Clamp Techniques; Picrotoxin; Rana pipiens; RNA, Messenger; Stilbenes

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K(+) efflux and Cl(-) loss. When human erythrocytes were suspended in a physiological NaCl solution (pH(o) = 7.4), the measured pH(i) was 7.19 + or - 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH(i) to 7.70 + or - 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl(-) content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H(+) influx with the measured unidirectional K(+) efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H(+) influx and a K(+) efflux in LIS solution of 108.2 + or - 20.4 mmol (l(cells) hr)(-1) and 98.7 + or - 19.3 mmol (l(cells) hr)(-1), respectively. For bovine and porcine erythrocytes, in LIS media, H(+) influx and K(+) efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K(+)(Na(+))/H(+) exchanger, inhibited the K(+) efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K(+)(Na(+))/H(+) exchanger in the human erythrocyte membrane.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anion Exchange Protein 1, Erythrocyte; Anions; Bumetanide; Cattle; Chlorides; Egtazic Acid; Erythrocyte Membrane; Extracellular Space; Female; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen; Hydrogen-Ion Concentration; Intracellular Fluid; Ion Channel Gating; Ion Transport; Male; Mitochondria; Niflumic Acid; Nigericin; Osmolar Concentration; Ouabain; Potassium; Quinacrine; Sodium; Sodium-Hydrogen Exchangers; Species Specificity; Stilbenes; Swine

We investigated whether volume-regulated anion channels (VRACs) contributed to the accumulation of extracellular adenosine during hypoxia in area CA1. The rapid hypoxic depression of the fEPSP was greatly attenuated by the selective adenosine A1 receptor antagonist DPCPX (50 nM), but not affected by the VRAC blockers tamoxifen (10-30 microM) or DNDS (1 mM). Our data argue against the efflux of adenosine per se or its precursor ATP through VRACs as making a significant contribution to extracellular adenosine during the early stages of hypoxia.

Topics: Adenosine; Adenosine Triphosphate; Animals; Anions; Estrogen Antagonists; Excitatory Postsynaptic Potentials; Female; Hippocampus; Hypoxia, Brain; In Vitro Techniques; Male; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Stilbenes; Synaptic Transmission; Tamoxifen; Xanthines

Serous cells secrete Cl(-) and HCO(3)(-) and play an important role in airway function. Recent studies suggest that a Cl(-)/HCO(3)(-) anion exchanger (AE) may contribute to Cl(-) secretion by airway epithelial cells. However, the molecular identity, the cellular location, and the contribution of AEs to Cl(-) secretion in serous epithelial cells in tracheal submucosal glands are unknown. The goal of the present study was to determine the molecular identity, the cellular location, and the role of AEs in the function of serous epithelial cells. To this end, Calu-3 cells, a human airway cell line with a serous-cell phenotype, were studied by RT-PCR, immunoblot, and electrophysiological analysis to examine the role of AEs in Cl(-) secretion. In addition, the subcellular location of AE proteins was examined by immunofluorescence microscopy. Calu-3 cells expressed mRNA and protein for AE2 as determined by RT-PCR and Western blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat tracheal serous cells in situ. In Cl(-)/HCO(3)(-)/Na(+)-containing media, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (I(sc)) was reduced by basolateral but not by apical application of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (50 microM) and 4, 4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 microM)], inhibitors of AEs. In the absence of Na(+) in the bath solutions, to eliminate the contributions of the Na(+)/HCO(3)(-) and Na(+)/K(+)/2Cl(-) cotransporters to I(sc), CPT-cAMP stimulated a small DNDS-sensitive I(sc). Taken together with previous studies, these observations suggest that a small component of cAMP-stimulated I(sc) across serous cells may be referable to Cl(-) secretion and that uptake of Cl(-) across the basolateral membrane may be mediated by AE2.

Topics: Animals; Anion Transport Proteins; Antiporters; Blotting, Western; Cell Line; Chloride-Bicarbonate Antiporters; Chlorides; Cyclic AMP; Electrophysiology; Enzyme Inhibitors; Epithelial Cells; Humans; Immunohistochemistry; Ion Transport; Male; Membrane Proteins; Protein Isoforms; Rats; Rats, Wistar; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serous Membrane; SLC4A Proteins; Stilbenes; Thionucleotides; Trachea

Muscle-stripped piglet colon was used to evaluate changes in short-circuit current (I(sc)) as an indicator of anion secretion. Mucosal exposure to Escherichia coli heat-stable (STa) or heat-labile enterotoxins (LT) stimulated I(sc) by 32 +/- 5 and 42 +/- 7 microA/cm(2), respectively. Enterotoxin-stimulated I(sc) was not significantly affected by either 4,4'-diaminostilbene-2, 2'-disulfonic acid or CdCl(2), inhibitors of Ca(2+)-activated Cl(-) channels and ClC-2 channels, respectively. Alternatively, N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), a compound that inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion, reduced I(sc) by 29 +/- 7 and 34 +/- 11 microA/cm(2), respectively. Two additional diarylsulfonylurea (DASU)-based compounds were evaluated for their effects on enterotoxin-stimulated secretion. The rank order of potency for inhibition by these three closely related DASU structures was identical to that observed for human CFTR. The degree of inhibition by each of these compounds was similar for both STa and LT. The structure- and concentration-dependent inhibition shown indicates that CFTR mediates both STa- and LT-stimulated colonic secretion. Similar structure-dependent inhibitory effects were observed in forskolin-stimulated rat colonic epithelium. Thus DASUs compose a family of inhibitors that may be of therapeutic value for the symptomatic treatment of diarrhea resulting from a broad spectrum of causative agents across species.

Topics: Amiloride; Animals; Anions; Antineoplastic Agents; Benzofurans; Biological Transport; Bumetanide; Cadmium Chloride; Colon; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Diuretics; Enterotoxins; In Vitro Techniques; Intestinal Mucosa; Phenylurea Compounds; Stilbenes; Sulfonylurea Compounds; Swine; Tetrodotoxin; Urea; Vasoactive Intestinal Peptide

A permanent cell line with inducible expression of the human anion exchanger protein 1 (hAE1) was constructed in a derivative of human embryonic kidney cells (HEK-293). In the absence of the inducer, muristerone A, the new cell line had no detectable hAE1 protein by Western analysis or additional 36Cl flux. Increasing dose and incubation time with muristerone A increased the amount of protein (both unglycosylated and glycosylated). The 4,4'-dinitrostilbene-2, 2'-disulfonate (DNDS)-inhibitable rapid Cl exchange flux was increased up to 40-fold in induced cells compared with noninduced cells. There was no DNDS-inhibitable rapid flux component in noninduced cells. This result demonstrates inducible expression of a new rapid Cl transport pathway that is DNDS sensitive. The additional transport of 36Cl and 35SO4 had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 microM DNDS, 2) activation of 36Cl efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3) activation of 36Cl efflux by external anions that was selective in the order NO3 = Cl > Br > I, and 4) activation of 35SO4 influx by external protons. Under the assumption that the turnover numbers of hAE1 were the same as in erythrocytes, there was good agreement (+/-3-fold) between the number of copies of glycosylated hAE1 and the induced tracer fluxes. This is the first expression of hAE1 in a mammalian system to track the kinetic characteristics of the native protein.

Topics: Anion Exchange Protein 1, Erythrocyte; Biological Transport; Blotting, Western; Cell Line; Chlorides; Ecdysterone; Fluorescent Antibody Technique; Humans; Stilbenes

Increased activation of excitatory amino acid (EAA) receptors is considered a major cause of neuronal damage. Possible sources and mechanisms of ischemia-induced EAA release were investigated pharmacologically with microdialysis probes placed bilaterally in rat striatum.. Forebrain ischemia was induced by bilateral carotid artery occlusion and controlled hypotension in halothane-anesthetized rats. During 30 minutes of ischemia, microdialysate concentrations of glutamate and aspartate were measured in the presence of a nontransportable blocker of the astrocytic glutamate transporter GLT-1, dihydrokinate (DHK), or an anion channel blocker, 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS), administered separately or together through the dialysis probe.. In control striata during ischemia, glutamate and aspartate concentrations increased 44+/-13 (mean+/-SEM) times and 19+/-5 times baseline, respectively, and returned to baseline values on reperfusion. DHK (1 mmol/L in perfusate; n=8) significantly attenuated EAA increases compared with control (glutamate peak, 9. 6+/-1.7 versus control, 15.4+/-2.6 pmol/ microL). EAA levels were similarly decreased by 10 mmol/L DHK. DNDS (1 mmol/L; n=5) also suppressed EAA peak increases (glutamate peak, 5.8+/-1.1 versus control, 10.1+/-0.7 pmol/ microL). At a higher concentration, DNDS (10 mmol/L; n=7) further reduced glutamate and aspartate release and also inhibited ischemia-induced taurine release. Together, 1 mmol/L DHK and 10 mmol/L DNDS (n=5) inhibited 83% of EAA release (glutamate peak, 2.7+/-0.7 versus control, 10.9+/-1.2 pmol/ microL).. These findings support the hypothesis that both cell swelling-induced release of EAAs and reversal of the astrocytic glutamate transporter are contributors to the ischemia-induced increases of extracellular EAAs in the striatum as measured by microdialysis.

Topics: Amino Acid Transport System X-AG; Animals; Aspartic Acid; ATP-Binding Cassette Transporters; Biological Transport; Blood Flow Velocity; Brain Ischemia; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Corpus Striatum; Drug Therapy, Combination; Glutamic Acid; Ion Pumps; Kainic Acid; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Stilbenes

1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradiol 17-(beta-D-glucuronide) (E217betaG) caused a voltage-dependent block of macroscopic CFTR Cl- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96+/-10 and 563+/-103 microM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453+/-44 and 3760+/-710 microM (at 0 mV) respectively. 3. Reducing the extracellular Cl- concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54+/-1 microM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane.

Topics: Animals; ATP-Binding Cassette Transporters; Calcium Channel Blockers; Chlorides; CHO Cells; Cholates; Cricetinae; Cystic Fibrosis Transmembrane Conductance Regulator; Dose-Response Relationship, Drug; Electric Stimulation; Estradiol; Glyburide; Membrane Potentials; Multidrug Resistance-Associated Proteins; ortho-Aminobenzoates; Stilbenes; Taurocholic Acid; Taurolithocholic Acid

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.

Topics: Benzimidazoles; Bicarbonates; Bumetanide; Calcium Channel Agonists; Cell Line; Chlorides; Colforsin; Diuretics; Electrophysiology; Epithelial Cells; Humans; Ion Channel Gating; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Stilbenes

The effects of pH on cotransporter kinetics were studied in renal proximal tubule cells. Cells were grown to confluence on permeable support, mounted in an Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, dinitrostilbene-disulfonate-sensitive current (DeltaI) was Na+ and HCO3- dependent and therefore was taken as flux through the cotransporter. When the pH of the perfusing solution was changed between 6.0 and 8.0, the conductance attributable to the cotransporter showed a maximum between pH 7.25 and pH 7.50. A similar profile was observed in the presence of a pH gradient when the pH of the apical solutions was varied between 7.0 and 8.0 (basal pH lower by 1), but not when the pH of the basal solution was varied between 7.0 and 8.0 (apical pH lower by 1 unit). To delineate the kinetic basis for these observations, DeltaI-voltage curves were obtained as a function of Na+ and HCO3- concentrations and analyzed on the basis of a kinetic cotransporter model. Increases in pH from 7.0 to 8.0 decreased the binding constants for the intracellular and extracellular substrates by a factor of 2. Furthermore, the electrical parameters that describe the interaction strength between the electric field and substrate binding or charge on the unloaded transporter increased by four- to fivefold. These data can be explained by a channel-like structure of the cotransporter, whose configuration is modified by intracellular pH such that, with increasing pH, binding of substrate to the carrier is sterically hindered but electrically facilitated.

Topics: Animals; Bicarbonates; Biophysical Phenomena; Biophysics; Carrier Proteins; Cell Line; Hydrogen-Ion Concentration; Ion Channel Gating; Ion Transport; Kidney Tubules, Proximal; Kinetics; Models, Biological; Rats; Sodium; Sodium-Bicarbonate Symporters; Stilbenes

As shown in earlier work (M.M. Henszen et al., Mol. Membr. Biol. 14 (1997) 195-204), exposure of erythrocytes to single brief electric field pulses (5-7 kV cm(-1)) enhances the transbilayer mobility of phospholipids and produces echinocytes which can subsequently be transformed into stomatocytes in an ATP-dependent process. These shape transformations arise from partly reversible changes of the transbilayer disposition of phospholipids, in agreement with the bilayer couple concept. Extensive membrane modification by repetitive (

Topics: Cell Size; Electroporation; Erythrocyte Deformability; Erythrocyte Membrane; Erythrocytes; Glutathione; Humans; Lipid Peroxidation; Membrane Proteins; Phospholipids; Pyrimidinones; Serum Albumin; Stilbenes; Temperature; Time Factors; Trypsin

A new metabotropic glutamate receptor (mGluR) agonist, (2S,1'S,2'S)-2-(2-carboxy-3,3-difluorocyclopropyl)glycine (L-F2CCG-I), induces a priming effect on (RS)-alpha-aminopimelate in the isolated spinal cord of newborn rats. Similar to (RS)-alpha-aminopimelate, L-glutamate (30-100 microM) neither affected spinal reflexes nor the resting membrane potentials of motoneurones, but preferentially potentiated the depression of monosynaptic excitation caused by L-F2CCG-I (0.4 microM). Following L-F2CCG-I treatment (1-2 microM), L-glutamate decreased the monosynaptic spinal reflexes in a concentration dependent manner, indicating a priming' effect of L-F2CCG-I. Thus L-glutamate is completely compatible with (RS)-alpha-aminopimelate in revealing the priming effect. An anion transport blocker, 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) (100 microM), markedly inhibited both the response to (RS)-alpha-aminopimelate and the induction of the L-F2CCG-I priming effect. The data suggest that L-F2CCG-I is Cl- -dependently incorporated into certain stores, and that (RS)-alpha-aminopimelate or L-glutamate must stimulate the release of L-F2CCG-I from the storage site. There were pharmacological similarities between the quisqualate and L-F2CCG-I priming effect. The physiological significance of the quisqualate or L-F2CCG-I priming is not yet established. L-F2CCG-I would be expected to be a useful pharmacological probe for elucidating the mechanism of the priming.

Topics: Amino Acids, Dicarboxylic; Animals; Biological Transport; Excitatory Amino Acid Agonists; Glutamic Acid; In Vitro Techniques; Membrane Potentials; Motor Neurons; Pimelic Acids; Quisqualic Acid; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Spinal Cord; Stilbenes; Synapses

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.

Topics: Amino Acid Sequence; Antiporters; Binding Sites; Chloride-Bicarbonate Antiporters; Cysteine; Erythrocyte Membrane; Humans; In Vitro Techniques; Lipid Bilayers; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Stilbenes

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Calcium; Cell Line; Chloride Channels; Ciliary Body; Enzyme Inhibitors; Epithelial Cells; Niflumic Acid; Osmotic Pressure; Patch-Clamp Techniques; Phosphorylation; Protein Kinase Inhibitors; Rabbits; Second Messenger Systems; Stilbenes

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Antiporters; Chloride-Bicarbonate Antiporters; Chlorides; Dimerization; Erythrocytes; Humans; Membrane Proteins; Microsomes; Oocytes; Peptide Fragments; Precipitin Tests; Stilbenes; Xenopus

In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of x 40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- > or = Br- >> I- > or = F-. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at -160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2 delta NT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2 delta NT-injected oocytes (-5.9 +/- 0.4 microA), followed by ClC-2-injected oocytes (-4.3 +/- 0.6 microA), and smallest in water-injected oocytes (-0.2 +/- 0.2 microA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 +/- 0.2%) > ClC-2-injected oocytes (5.3 +/- 0.5%) > ClC-2 delta NT-injected oocytes (1.1 +/- 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopus oocytes appear to act for volume regulation when exposed to a hyposmolar environment.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cell Membrane Permeability; Chloride Channels; CLC-2 Chloride Channels; Colforsin; Electrophysiology; Nerve Tissue Proteins; Nitrobenzoates; Oocytes; Osmolar Concentration; Rabbits; Rats; Stilbenes; Water-Electrolyte Balance; Xenopus

1. The effect of membrane modification by the arginine-binding reagent phenylglyoxal (PG) on Cl- permeability was studied in thin bundles of twitch fibres from frog muscle. The bundles were modified by a method that yields stable PG binding to outer arginyl residues in erythrocyte membranes. 2. PG almost eliminated the pH-dependent fraction of 36Cl- efflux under conditions of Cl- equilibrium in depolarized bundles: the fluxes at pH 7.2 and 8.5 were strongly inhibited approaching an apparent baseline value close to the normal flux at pH 6 which per se was not inhibited. 3. The uninhibited flux at pH 6 in modified bundles maintained the normally high sensitivity to 4,4'-dinitro-stilbene-2,2'-disulphonate (DNDS), and the reduction of fluxes at pH > 7 coincided with increased DNDS sensitivity, suggesting a selective blocking of the pH-dependent flux fraction that has a low DNDS sensitivity. 4. In normal Ringer solution the modified fibres showed normal resting membrane potentials (Vm) with normal sensitivity to [K+]o but sensitivity to changes of [Cl-]o was almost eliminated, suggesting a normal resting Na+:K+ conductance ratio (gNa/gK) and that the main influence of modification on the resting membrane conductance (gm) was a loss of Cl- conductance (gCl). 5. The modified fibres were not excitable, possibly due to arginine modification in the voltage sensor (S4) of the Na+ channels. 6. These results suggest that positively charged arginines are important for the activity of the pH-dependent Vm-stabilizing Cl- channels and that PG may isolate a pH-independent basal flux fraction which normally dominates the Cl- flux at low pH.

Topics: Animals; Arginine; Chloride Channels; Electric Stimulation; Electrophysiology; Enzyme Inhibitors; Erythrocyte Membrane; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Muscle Fibers, Skeletal; Muscle, Skeletal; Patch-Clamp Techniques; Phenylglyoxal; Rana temporaria; Stilbenes

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.

Topics: Animals; Calcium; Chloride Channels; Glycolates; In Vitro Techniques; Kinetics; Muscle, Smooth; Nitrobenzoates; Rabbits; Sarcoplasmic Reticulum; Stilbenes; Stomach

Phenylglyoxalation of the red blood cell membrane leads to three superimposed effects on band 3 protein-mediated anion equilibrium exchange as measured by means of radiosulfate: (1) a shift of the curve relating transport activity to pH towards lower pH values, possibly in combination with an increase of the maximal transport activity. This is accompanied by effect (2), the abolishment of a chloride-stimulated component of anion transport seen at low pH values. Effect (3) consists of inhibition of anion equilibrium exchange. Effect (1) prevails when phenylglyoxalation is performed at low concentrations of PG and low pH, while effect (3) predominates when exposure to PG is executed at high pH and high concentration of PG. Effect (1) is associated with a decrease of the Ki values for inhibition and binding of the reversibly acting stilbene disulfonates DNDS and DBDS. The inhibition observed as a consequence of effect (3) is linearly related to a decrease of the capacity of band 3 to combine with the stilbene disulfonate DBDS. The results are interpreted on the assumption that PG is capable of reacting with two or possibly three distinct binding sites in band 3. Reaction with one of them leads to effect (1) and, perhaps, to effect (2); reaction with the other to effect (3). The latter is possibly due to modification of Arg 730, which is homologous to Arg 748 in mouse band 3. Site-directed mutagenesis of this arginine residue showed that it is required for band 3-mediated anion transport.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Arginine; Binding Sites; Biological Transport; Chlorides; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Kinetics; Phenylglyoxal; Stilbenes; Sulfates

Taurine has been shown to act as an osmolyte during the regulatory volume decrease process in a variety of cell types. The nature of the taurine efflux carrier is thought to consist of a diffusional pathway with pharmacological properties similar to a chloride channel or through an anion exchanger. We propose that taurine is a substrate of the anion exchanger AE1, also called band 3. Experiments were performed in rat-erythrocytes, which express large amounts of band 3. Taurine uptake and efflux transport experiments were determined in the presence of inhibitors of anion carriers and chloride channels. Both taurine uptake and efflux were inhibited by band 3 inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), niflumic acid, or furosemide. Moreover, DIDS competes with taurine at a common binding site in the uptake process. Specific inhibitors of the electroneutral cotransport as well as inhibitors of the chloride channels were ineffective in blocking taurine transport. Thus we suggest that band 3 may be the protein responsible for taurine transport in rat erythrocytes.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Exchange Protein 1, Erythrocyte; Antiporters; Binding Sites; Binding, Competitive; Biological Transport; Bumetanide; Chloride Channels; Erythrocytes; Rats; Rats, Sprague-Dawley; Stilbenes; Taurine

Dimeric Band 3 purified in n-dodecyl octaethyleneglycol (C12E8) underwent an irreversible, temperature-dependent association, resulting in a complex with a Stokes radius slightly larger than a native tetramer, before forming a higher molecular weight aggregate. Self-association occurred with a half-time of about 1 h at 37 degrees C but did not occur at 0 degrees C after several days. No change in the secondary structure of Band 3, as observed by circular dichroism, occurred during the association process. However, self-association of Band 3 was accompanied by loss of the stilbene disulfonate inhibitor binding site. No association or loss of inhibitor binding occurred with the dimeric membrane domain under similar incubation conditions. The membrane domain dimer was also stable over a wide range of pH (5.5-9.5) and buffer conditions, while Band 3 aggregated below pH 6.5. Inhibitors of anion transport, which stabilize the membrane domain, slowed the association. Band 3, depleted of phospholipids by extensive washing of resin-bound protein with detergent or, incubated with excess detergent, was more prone to aggregation. The membrane domain also showed some aggregation when depleted of lipids. Preparations could be stabilized by adding dimyristoylphosphatidylcholine (DMPC) prior to the 37 degrees C incubation. The effect of inhibitors and DMPC was additive, with a combination of 1 mM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) and 1:1 (wt/wt) DMPC:Band 3 stabilizing 90% of the protein to a 24-h incubation at 37 degrees C. The results suggest that self-association of Band 3 dimers is promoted by the cytoplasmic domain but results in alterations to the membrane domain involving the loss of essential phospholipids. Addition of phospholipid or inhibitors to Band 3 results in a stable preparation of the intact protein that may be suitable for crystallization studies.

Topics: Anion Exchange Protein 1, Erythrocyte; Chromatography, Gel; Chromatography, High Pressure Liquid; Detergents; Dimerization; Dimyristoylphosphatidylcholine; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Protein Structure, Secondary; Stilbenes; Temperature; Time Factors

Human erythrocytes are known to reduce ferricyanide (hexacyanoferrate) [Fe(CN)6]3- to ferrocyanide [Fe(CN)6]2- in an extracellular reaction that involves the transmembrane transfer of reducing equivalents; potentially these could be either electrons from NADH, formed in glycolysis inside the cells or transmembrane exchange of reduced solutes. The 13C-NMR resonance of [Fe(13CN)6]3- (which was synthesised in our laboratory) was seen to be very broad while that of ferrocyanide was narrow. This phenomenon formed the basis of a simple non-invasive procedure to study ferricyanide reduction in high-haematocrit suspensions of erythrocytes. The method should be directly applicable to other cell types. In a series of experiments, erythrocyte metabolism was studied in the presence of ferricyanide, using 1H, 13C, and 31P NMR spectroscopy. Incubating the cells with 13C-labelled glucose enabled the rate of ferricyanide reduction, glucose utilisation, and lactate and bicarbonate production to be measured simultaneously. Various metabolic states were imposed as follows: glycolysis was inhibited with F- and iodoacetate; glucose transport was inhibited with phloretin and cytochalasin B; and anion transport was inhibited with dinitrostilbene 2,2'-disulfonate and p-chloromercuriphenyl sulfonate. Earlier work was confirmed, showing that ascorbate is intimately involved in the reduction reaction; but its main action appears not to be mediated by membrane transport but in a membrane-associated redox-protein complex that is functionally linked to glycolysis. Also, large differences (factors of three) in the rate of the reduction reaction were recorded in erythrocytes from different, apparently healthy, donors.

Topics: 4-Chloromercuribenzenesulfonate; Ascorbic Acid; Biological Transport; Carbon Isotopes; Cytochalasin B; Dehydroascorbic Acid; Deoxyglucose; Electron Transport; Enzyme Inhibitors; Erythrocytes; Extracellular Space; Ferricyanides; Fluorides; Glucose; Humans; Iodoacetates; Iodoacetic Acid; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Phloretin; Stilbenes

The ability of a Cl-secreting epithelium to support net secretion of an anion other than a halide was investigated with 35SO4 flux measurements across the isolated, short-circuited rabbit distal colon. In most experiments, 36Cl fluxes were simultaneously measured to validate the secretory capacity of the tissues. Serosal addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mM) stimulated a sustained net secretion of SO4 (about -3.0 nmol.cm-2.h-1 from a 0.20 mM solution) via an increase in the serosal-to-mucosal unidirectional flux, whereas Ca ionophore A-23187 (1 microM, serosal) produced a more transient stimulation of SO4 and Cl secretion. Net adenosine 3',5'-cyclic monophosphate (cAMP)-dependent SO4 and Cl secretion were strongly voltage sensitive, principally through the potential dependence of the serosal-to-mucosal fluxes, indicating an electrogenic transport process. Symmetrical replacement of either Na, K, or Cl inhibited cAMP-dependent SO4 secretion, whereas HCO3-free buffers had no effect on SO4 secretion. Serosal bumetanide (50 microM) or furosemide (100 microM) reduced DBcAMP-stimulated SO4 and Cl secretion, whereas serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (50 microM) blocked DBcAMP-induced SO4 secretion while enhancing net Cl secretion and short-circuit current. Mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid partially inhibited SO4 secretion and completely inhibited Cl secretion. It is concluded that secretagogue-stimulated SO4 secretion, like Cl secretion, may be an electrogenic process mediated by diffusive efflux through an apical anion conductance. Cellular accumulation of SO4 across the basolateral membrane appears to be achieved by a mechanism that is distinct from that employed by Cl.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Biological Transport, Active; Bucladesine; Calcimycin; Chloride Channels; Chlorides; Colon; Cross-Linking Reagents; Cyclic AMP; Electrolytes; Female; In Vitro Techniques; Intestinal Mucosa; Kinetics; Male; Nitrobenzoates; Rabbits; Stilbenes; Sulfates

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 +/- 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl- than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at -20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br- > Cl- > I- approximately F-. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br- and Cl- have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 microM DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl-. Our results suggest that channel block involves the interaction of HPO2-4 with a site located at 70% of the membrane span.

Topics: Animals; Calcium; Chloride Channels; Endoplasmic Reticulum, Rough; Hydrogen; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Lipid Bilayers; Liver; Nitrobenzoates; Phosphates; Potassium; Rats; Stilbenes

Although many polarized proteins are sorted to the same membrane domain in all epithelial tissues, there are some that exhibit a cell type-specific polarity. We recently found that band 3 (the anion exchanger AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low density, but its targeting was reversed to the basolateral membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high density. Because apical and basolateral lipids differ in epithelia, we asked what effect might these lipids have on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant to disulfonic stilbene inhibitors while basolateral anion exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that readily identify the basolateral protein. We used short chain sphingolipid analogues and found that sphingomyelin was preferentially targeted to the basolateral domain in the intercalated cell line. The ganglioside GM1 (Gal 1beta1, 3GalNAcbeta1, 4Gal-NeuAcalpha2, 3Galbeta1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after addition of fluorescent cholera toxin to filter grown cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and examined the inhibitory potency of 4, 4'-dinitorsostilbene-2,2'-disulfonic acid (DNDS; a reversible stilbene) on 35SO4/SO4 exchange. Although anion exchange in sphingomyelin liposomes was sensitive to inhibition, the addition of increasing amounts of the ganglioside GM1 reduced the potency of the inhibitor drastically. Because these polarized lipids are present in the exofacial surface of the bilayer, we propose that the lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing the function of unpolarized proteins or of proteins whose locations differ in different epithelia.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Biological Transport; Cell Compartmentation; Cell Polarity; Cells, Cultured; Ceramides; Hydrogen-Ion Concentration; Kidney; Kinetics; Membrane Lipids; Proteolipids; Rabbits; Stilbenes; Sulfates

Transient currents are activated by spontaneous Ca2+ oscillations in rabbit ventricular myocytes. We investigated the ionic basis for these transient currents under conditions in which K+ currents would be expected to be blocked. Holding cells under voltage clamp at positive potentials leads to a rise in intracellular Ca2+ via reversal of the Na+-Ca2+ exchanger and subsequently to the initiation of spontaneous Ca2+ transients, presumably from a Ca2+-overloaded sarcoplasmic reticulum. The current transients associated with these Ca2+ transients reversed at about +10 to +15 mV under conditions of approximately symmetrical Cl-. In the absence of Cl-, this current was inward at all potentials examined over the range from -88 to +72 mV, consistent with a Na+-Ca2+ exchanger current. In the absence of Na+, the repetitive spontaneous Ca2+ transients could be initiated by a brief train of depolarizations to activate the inward Ca2+ current. Under such conditions, the current was found to reverse at -3 mV when the equilibrium potential of Cl- (ECl) was -2 mV, and the reversal potential shifted to -32 mV when internal Cl- was lowered, to make ECl -33 mV. Thus, in the absence of Na+, it appears that the current is exclusively a Ca2+-activated Cl- current. There is no evidence to indicate the presence of a Ca2+-activated cationic conductance. Further, our results demonstrate that the Ca2+-activated Cl- conductance can carry inward current at potentials more negative to ECl in rabbit ventricular myocytes and is therefore likely to contribute to the arrhythmogenic delayed afterdepolarizations that occur in Ca2+-overloaded cells.

Topics: Animals; Calcium; Calcium Channels; Carrier Proteins; Chlorides; Electric Conductivity; Heart Ventricles; Intracellular Membranes; Myocardium; Oscillometry; Patch-Clamp Techniques; Rabbits; Sodium-Calcium Exchanger; Stilbenes; Ventricular Function

The effects of 4-hydroxy-3-nitrophenylglyoxal (HNPG), on the binding of eosin-5-maleimide (EMA), and diethyl pyrocarbonate (DEPC) to the anion transport system in the human red blood cell membrane, have been investigated. HNPG is a reversibly binding, arginine-specific, anion transport competitive inhibitor, known to act on the anion binding site. The EMA reaction site is an external facing lysine residue (Lys-430) in the 17 kDa transmembrane segment. The DEPC reaction site is an intracellular histidine (His-819) in the 35 kDa fragment. The results show that inhibition of the transport system with EMA increases in presence of HNPG to about 2.3 times. This finding suggests a positive cooperativity between the HNPG and EMA binding site and give evidence that the essential arginine is either nearby or allosterically linked to Lys-430. The inhibition of the cells with DEPC was nearly unchanged or slightly decreased in the presence of 10 mM HNPG. These results suggest that the intracellular His-residue which reacts with DEPC is not a part of the transport pathway. Our experiments with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) have shown that its affinity to the transport system does not change after pre-treatment with phenylglyoxal (PG). We also found that the binding of [14C]phenylglyoxal (PG) to band 3 reduces significantly in presence of chloride. This is another evidence for the direct involvement of arginine residues in substrate binding.

Topics: Anion Exchange Protein 1, Erythrocyte; Arginine; Carbon Radioisotopes; Diethyl Pyrocarbonate; Erythrocyte Membrane; Humans; Phenylglyoxal; Protein Binding; Stilbenes

Caenorhabditis elegans spermatogenesis is especially suited for studies of nonrandom cytoplasmic segregation during cellular differentiation. Spermatocytes separate from an anuclear cytoplasmic core and undergo two sequential divisions. During the second division, intracellular organelles segregate specifically to spermatids as they bud from an anuclear residual body. We have applied patch-clamp techniques in order to investigate membrane protein distribution during these asymmetric divisions. We show that membrane components, as assayed by voltage-dependent ion channel activity, follow a specific distribution pattern during sperm development. Several voltage-sensitive ion channel activities are observed in spermatocytes and residual bodies, but only a single-channel type can be detected in spermatids, indicating that other channel activities are excluded from or inactivated within these cells as they form. The channel that is observed in spermatids is an inward-rectifying chloride channel (Clir), as indicated by its sensitivity to chloride channel inhibitors and Cl-dependent shifts in its conductance. Treatment of spermatids with Cl channel blockers induce their differentiation into spermatozoa, suggesting that Clir plays a role during this developmental step. These studies are the first application of patch-clamp electrophysiology to C. elegans development.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Caenorhabditis elegans; Cell Differentiation; Chloride Channels; Chlorides; Electrophysiology; Glycolates; Male; Nitrobenzenes; Patch-Clamp Techniques; Potassium; Pronase; Sodium; Spermatids; Spermatogenesis; Spermatozoa; Stilbenes

Volume expansion of little skate (Raja erinacea) erythrocytes increases the affinity of ankyrin binding without altering in the number of binding sites. Potassium iodide-stripped inside-out vesicles (KI-IOV) were used to assess ankyrin binding under volume-expanded conditions. Under isoosmotic conditions, ankyrin binds nearly exclusively to a single class of sites (Bmax, 52 +/- 12 microg/mg; Kd, 150 +/- 28 nM). KI-IOV from volume-expanded cells (either with one-half osmolarity medium or with inclusion of the permeant solute ethylene glycol) demonstrate two ankyrin-binding populations. A high affinity population occurs transiently under volume-expanded conditions. This population has a Bmax of 18 +/- 7 microg/mg and a Kd of 25 +/- 9 nM. Total binding of high and low affinity sites is 57 +/- 17 microg/mg. This change in ankyrin affinity is reversible on volume regulatory decrease. A major target protein in the KI-IOV was identified as the skate homolog of the mammalian red cell anion exchanger band 3. Inclusion of the purified cytoplasmic domain of band 3 competes away more than 80% of the ankyrin binding. To determine whether increased ankyrin affinity is due to band 3 tetramer formation that occurs in volume expansion, cells were treated with pyridoxal 5-phosphate or 4,4'-dinitrostilbene-2,2'-disulfonic acid, two agents that increase tetramer formation under isoosmotic conditions. Both treatments altered the binding affinity with a shift toward higher affinity binding without significant alteration in the number of binding sites.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Ankyrins; Binding Sites; Erythrocyte Volume; Protein Binding; Pyridoxal Phosphate; Skates, Fish; Stilbenes

1. Inhibitory postsynaptic potentials (IPSPs) evoked by stimulation at the white-matter/layer VI border were recorded intracellularly from visual cortical layer IV neurons maintained in vitro. These IPSPs, typically not apparent at resting membrane potentials, were measured at membrane potentials 15-25 mV depolarized from resting levels. The effects of two chloride channel blockers on these IPSPs were investigated. 2. 4,4'-Dinitro-stilbene-2,2'-disulfonic acid (DNDS) was found to inhibit IPSPs as it diffused into the postsynaptic cell from an intracellular micropipette, leaving only the presumed pure excitatory postsynaptic potential (EPSP) component of the evoked compound PSP. Input resistance, resting membrane potential, spike accommodation, and EPSPs at resting membrane potentials were not significantly affected. 3. A novel chloride channel blocker 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]a rene (TS-TM-calix[4]arene) was found to potently inhibit IPSPs recorded at depolarized membrane potentials. The TS-TM-calix[4]arene, similar to DNDS, did not affect input resistance, resting membrane potential, spike accommodation, and EPSPs at resting membrane potentials. 4. To confirm that DNDS and TS-TM-calix[4]arene were indeed blocking IPSPs, similar experiments were performed on monosynaptic IPSPs evoked by stimulation of layer V in the presence of 2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both DNDS and TS-TM-calix[4]arene were effective in blocking monosynaptic IPSPs. 5. Consistent with the notion that DNDS and TS-TM-calix[4]arene block IPSPs by inhibiting gamma-aminobutyric acid-A (GABAA) receptor channels, the decrease in input resistance caused by extracellular application of muscimol was also significantly inhibited by intracellular use of these compounds. 6. These data suggest that DNDS and TS-TM-calix[4]arene applied intracellularly may be useful for the study of the function of GABAA-mediated synaptic inhibition of cortical neurons. Because only neurons impaled by the recording electrodes are influenced by the drugs, this method offers an advantage over extracellular application of GABAA blockers in that entire networks of neurons are not influenced.

Topics: Animals; Calcium Channel Blockers; Chloride Channels; Electric Stimulation; Evoked Potentials; GABA-A Receptor Antagonists; Guinea Pigs; In Vitro Techniques; Membrane Potentials; Neurons; Receptors, Neurotransmitter; Stilbenes; Visual Cortex

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the substrate and inhibitor specificity of monocarboxylate transport into isolated rat heart cells. Further evidence was obtained for the presence of two lactate carriers present in heart cells (Wang et al., Biochem. J. 290: 249-258, 1993) both distinct from the recently cloned monocarboxylate transporter isoform 1 (MCT-1) found in many other cell types. Only one isoform was potently inhibited by alpha-cyano-4-hydroxycinnamate [CHC; inhibitor constant (Ki) 190 microM] and the stilbene disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki 79 microM) and 4,4'-dinitrostilbene-2,2'-disulfonate (Ki of cis- and trans-isomers 38 and 171 microM, respectively; neither isomer inhibits MCT-1). The second carrier had a Ki of approximately 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues, in rat heart cells these inhibitors are not effective at blocking lactate transport totally unless used at very high concentrations. Both carriers were inhibited by 3-isobutyl-1-methylxanthine (Ki 340 microM) and neither by 5-nitro-2-(3-phenylpropylamino)benzoate (a potent inhibitor of MCT-1). The overall Michaelis constant (Km) and maximum reaction rate (Vmax) for transport of a variety of substituted monocarboxylates (C2-C5) were determined, although it was not possible to elucidate the kinetic parameters of the two isoforms. Of physiological interest, the ketone bodies D-beta-hydroxybutyrate and acetoacetate had K(m) values of 10 and 5.4 mM, respectively. Vmax values were similar to those of L-lactate and pyruvate and indicate that transport could limit rates of utilization of ketone bodies. No stereoselectivity for L-over D-isomers of 2-chloro or 2-hydroxy acids was observed.

Topics: 1-Methyl-3-isobutylxanthine; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Carrier Proteins; Cell Separation; Cinnamates; Coumaric Acids; Fluoresceins; Fluorescent Dyes; Isomerism; Lactic Acid; Monocarboxylic Acid Transporters; Myocardium; Rats; Stilbenes; Substrate Specificity

We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.

Topics: Anion Exchange Protein 1, Erythrocyte; Binding Sites; Biophysical Phenomena; Biophysics; Chlorides; Chlorine; Eosine Yellowish-(YS); Humans; In Vitro Techniques; Ion Transport; Magnetic Resonance Spectroscopy; Stilbenes

The aim of this study was to determine the effects of Cl- channel blockers on the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (ICl) and the protein kinase A-regulated L-type calcium current (PKA-ICa).. Whole-cell ICl and ICa were recorded from isolated guinea pig ventricular myocytes using the patch clamp technique during stimulation of PKA by forskolin (1 or 2 microM).. The inhibitory effects of clofibric acid, p-chlorophenoxy propionic acid, gemfibrozil, diphenylamine-2-carboxylate (DPC), anthracene-9-carboxylate, 4,4'dinitrostilbene-2,2'-disulfonic acid and indanyloxyacetic acid 94 were examined on the two currents. Clofibric acid (1 mM), p-chlorophenoxy propionic acid (1 mM) and gemfibrozil (250 microM) produced an approximate 50% decrease in ICl, but had no effect on the PKA-ICa. Surprisingly, application of DPC (500 microM and 1 mM) and anthracene-9-carboxylate (500 microM) strongly reduced both currents. However, inhibition of the Ca2+ and Cl- channels by DPC could be differentiated in two important ways. First, increasing the pH of the external solution from 7.4 to 10.0 prevented the block of ICl by DPC, but did not attenuate the reduction in the PKA-ICa. Second, DPC inhibited the PKA-ICa in mouse atrial myocytes which lacked ICl. Neither 4,4'dinitrostilbene-2,2'-disulfonic acid (100 microM) nor indanyloxyacetic acid 94 (50 microM) caused any change in either of the guinea pig ventricular currents.. Drugs such as DPC and anthracene-9-carboxylate which block the cardiac CFTR Cl- channel also inhibit the regulation of the L-type ICa. During beta-adrenergic stimulation, changes produced by these drugs on the cardiac action potential duration will be attributable to inhibition of both the Cl- and Ca2+ currents. Analogues of clofibric acid may serve as selective blockers of the CFTR Cl- channel that can be used to determine the physiological function of ICl in cardiac excitation.

Topics: Animals; Anthracenes; Calcium; Calcium Channel Blockers; Chloride Channels; Chlorides; Clofibric Acid; Colforsin; Cystic Fibrosis Transmembrane Conductance Regulator; Fenofibrate; Gemfibrozil; Glycolates; Guinea Pigs; Ion Transport; Mice; Myocardium; ortho-Aminobenzoates; Patch-Clamp Techniques; Stilbenes

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.

Topics: Amino Acids; Anion Exchange Protein 1, Erythrocyte; Binding, Competitive; Chlorides; Cross-Linking Reagents; Humans; Hydrogen-Ion Concentration; Iodides; Kinetics; Stilbenes; Succinimides; Sulfates

To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Body Fluids; Chlorides; Colforsin; Doxorubicin; Drug Resistance, Multiple; Electric Conductivity; Female; Humans; Patch-Clamp Techniques; Potassium; RNA, Messenger; Stilbenes; Tumor Cells, Cultured; Verapamil

1. The disulphonic stilbenes 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) were shown to cause a voltage-dependent inhibition of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in baby hamster kidney cells when applied to the cytoplasmic face of the membrane. These compounds are known to be relatively ineffective at blocking CFTR from the extracellular side of the membrane. 2. Mutation of a positively charged arginine, previously suggested to be located in the channel pore (R347), to a negatively charged aspartate significantly reduced the affinity of block by both DNDS and DIDS, suggesting that this residue contributes to the binding site for disulphonic stilbenes. 3. It is suggested that the CFTR Cl- channel may contain a relatively large inner vestibule in which a number of large anions bind and block Cl- permeation. Arginine 347 may be involved in anion binding within this region.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Arginine; Aspartic Acid; Binding Sites; Cell Line; Cell Membrane; Cricetinae; Cystic Fibrosis Transmembrane Conductance Regulator; Kidney; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins; Stilbenes; Transfection

Expression of voltage-activated ion channels was studied in primary cultures from seven freshly resected human primary brain tumors and in an established human astrocytoma cell line, STTG1. Astrocytoma cells consistently expressed voltage-dependent outwardly rectifying currents. Currents activated at potentials > 45 mV and showed outward transients on termination of voltage steps. Currents reversed at the Cl equilibrium potential, suggesting that they were largely carried by Cl-. Altering extracellular K- or Na+ concentration did not alter currents; neither did replacement of intracellular K+ by Cs+ or intracellular Na+ by N-methyl-D-glucosamine. Anion-substitution experiments suggest the following permeability sequence, determined from shifts in tail current reversal potential: I- > NO3- > Br- > Cl- > acetate > isethionate > F- > glutamate. Currents were sensitive to the Cl- channel blockers chlorotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 4,4'-dinitrostilbene-2,2' disulfonic acid (DNDS), with chlorotoxin being most effective, yielding > 80% block at 590 nM. DIDS (100 microM) and DNDS (100 microM) reduced currents by 33.5 and 38.2%, respectively. Currents were also sensitive to Zn2+ (100 microM, 47% block) and Cd2- (25 microM, 42% block). Reducing extracellular Ca2+ concentration decreased outward currents by 58% and almost completely eliminated transients, suggesting that Cl- currents are Ca2+ dependent. Cl channel block resulted in altered cell proliferation as determined by [3H]thymidine incorporation, suggesting that these channels may be involved in astrocytoma growth control.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Astrocytoma; Brain Neoplasms; Cell Division; Chloride Channels; Chlorides; Electric Conductivity; Humans; Scorpion Venoms; Stilbenes; Tumor Cells, Cultured

1. The effects of angiotensin II (Ang II) on membrane currents were investigated in single ventricular myocytes from the rabbit heart by the whole-cell voltage-clamp method. 2. In the presence of an inhibitor of Ca2+ currents (nifedipine at 3 microM or CdCl2 at 0.3 mM) and a beta-adrenoceptor blocker (bupranolol at 1 microM), 1 microM Ang II significantly increased the membrane conductance. 3. After elimination of K+ from external and internal solutions and its replacement by Cs+, Ang II at 0.1 microM increased an outwardly rectifying current that reached a maximum after about 40 min. The effect was concentration dependent (10(-9)-10(-6) M) and was inhibited by saralasin, an antagonist of Ang II receptors. 4. The reversal potential of the Ang II-induced current in the absence of K+ was compatible with the Cl- equilibrium potential at various external concentrations of Cl-. 5. A Cl- channel blocker, 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS, at 5 mM), reversibly decreased the Ang II-induced current. 6. The Ang II-induced current developed when the internal solution contained Ca2+ (pCa 7.2 or 7.0) but not when it contained 10 mM EGTA without Ca2+. 7. Besides developing a Cl- current, Ang II at 1 microM increased the inwardly rectifying K+ current (IK1) and this effect reached maximum within 3 min. 8. The effect of Ang II on the action potential was biphasic: the duration of the action potential was initially reduced and then it was increased. 9. These results suggest that Ang II induces a Cl- current that appears likely to modulate the action potential in rabbit ventricular myocytes.

Topics: Action Potentials; Angiotensin II; Animals; Bupranolol; Cadmium; Cadmium Chloride; Calcium; Chlorides; Egtazic Acid; Ion Channels; Kinetics; Male; Myocardium; Nifedipine; Patch-Clamp Techniques; Potassium; Rabbits; Receptors, Adrenergic, alpha-1; Saralasin; Stilbenes

Renal brush border membranes contain several anion exchanges that may play a role in electrolyte transport and pH regulation. To help characterize the types of exchangers present and the binding properties of these membranes, the binding of nitrate (NO3-) to highly purified rabbit kidney brush border membrane vesicles was studied. The method is based on the binding induced quadrupole relaxation of the 14N-NMR signal of nitrate [1,2]. Brush border membrane vesicles caused a relaxation of the 14N-NMR nitrate signal which could be characterized by relatively high affinity sites, KD = 6.7 +/- 1.5 mM, as well as nonspecific interactions with the membranes, KD > 150 mM. The anion transport inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) inhibited 51 +/- 6% (n = 4) of the relaxation due to the high affinity binding sites. The DNDS inhibition could be characterized by a Ki of 10-80 microM. Both bicarbonate and formate (HCO2-) were found to partially inhibit the high affinity induced relaxation, with maximal inhibition of 37 +/- 8% (n = 3) and 30 +/- 2% (n = 3), respectively. The inhibitory effects of saturating concentrations of bicarbonate and formate were non-additive, suggesting the existence of a stilbene sensitive exchanger that can bind nitrate, as well as both bicarbonate and formate. This study indicates the usefulness of this new method for further investigation of anion exchangers on these and other membranes.

Topics: Animals; Bicarbonates; Binding Sites; Carbonic Anhydrases; Formates; Kidney; Magnetic Resonance Spectroscopy; Microvilli; Nitrates; Nitrogen Isotopes; Rabbits; Stilbenes

The kinetic properties of sulfate transport mediated by the anion exchangers AE1 and AE2 have been examined. Microsomes isolated from HEK cells transiently overexpressing either protein were reconstituted in unilamellar, 200-600-nm diameter proteoliposomes. Transport mediated by the exchangers was monitored by loading the reconstituted proteoliposomes with the slowly transportable anion SO4(2-) using [35S]SO4(2-) as a tracer and performing [35S]SO4(2-)/SO4(2-) exchange. The following data suggest that AE1 and AE2 have been functionally reconstituted: (i) the rate of SO4(2-) transport in AE1 and AE2 containing proteoliposomes was 10-20 times higher than in proteoliposomes derived from control microsomes; (ii) the transport of SO4(2-) was strongly dependent on the presence of a trans anion; and (iii) the anion exchanger inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-di-sulfonate (DIDS) totally abolished SO4(2-) transport. furthermore, DIDS inhibits SO4(2-) transport only when occluded inside the vesicles, indicating a uniform, asymmetrical, inside-out orientation of the reconstituted exchangers. The Ki values of the stilbene disulfonate compound DNDS were 2.5 and 4 microM for AE1 and AE2, respectively, suggesting that the two exchangers possess similar high affinity sites for stilbene compounds. Both AE1 and AE2 showed the same steep pH dependence of sulfate transport, which was maximal at pH 5.5 and reduced to less than 10% (of the value at pH 5.5) at pH 8.5, suggesting that an acidic residue shared by AE1 and AE2 participates in the pH regulation of sulfate transport.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antiporters; Binding Sites; Cell Line; Humans; Hydrogen-Ion Concentration; Kidney; Kinetics; Liposomes; Mice; Microscopy, Electron; Microsomes; Proteolipids; Radioisotope Dilution Technique; Recombinant Proteins; Stilbenes; Sulfates; Sulfur Radioisotopes; Transfection

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.

Topics: Anion Exchange Protein 1, Erythrocyte; Binding Sites; Binding, Competitive; Biophysical Phenomena; Biophysics; Chlorides; Eosine Yellowish-(YS); Fluorescent Dyes; Hematocrit; Hemolysis; Humans; In Vitro Techniques; Iodides; Ion Channels; Ion Exchange; Magnetic Resonance Spectroscopy; Models, Chemical; Stilbenes

We have characterized a voltage-sensitive chloride channel from cardiac sarcoplasmic reticulum (SR) following reconstitution of porcine heart SR into planar lipid bilayers. In 250 mM KCl, the channel had a main conductance level of 130 pS and exhibited two substrates of 61 and 154 pS. The channel was very selective for Cl- over K+ or Na+ (PK+/PCl- = 0.012 and PNa+/PCl- approximately 0.040). It was permeable to several anions and displayed the following sequence of anion permeability: SCN- > I- > NO3- approximately Br- > Cl- > F- > HCOO-. Single-channel conductance saturated with increasing Cl- concentrations (Km = 900 mM and gamma max = 488 pS). Channel activity was voltage dependent, with an open probability ranging from approximately 1.0 around 0 mV to approximately 0.5 at +80 mV. From -20 to +80 mV, channel gating was time-independent. However, at voltages below -40 mV the channel entered a long-lasting closed state. Mean open times varied with voltage, from approximately 340 msec at -20 mV to approximately 6 msec at +80 mV, whereas closed times were unaffected. The channel was not Ca(2+)-dependent. Channel activity was blocked by disulfonic stilbenes, arylaminobenzoates, zinc, and cadmium. Single-channel conductance was sensitive to trans pH, ranging from approximately 190 pS at pH 5.5 to approximately 60 pS at pH 9.0. These characteristics are different from those previously described for Cl- channels from skeletal or cardiac muscle SR.

Topics: Animals; Anions; Chloride Channels; Electric Conductivity; Hydrogen-Ion Concentration; In Vitro Techniques; Ion Channel Gating; Lipid Bilayers; Membrane Potentials; Myocardium; Nitrobenzoates; Permeability; Sarcoplasmic Reticulum; Stilbenes; Swine; Zinc

Swelling-induced chloride currents may contribute to cardiac electrical activity and cell volume regulation. Identification of selective blockers would aid in understanding the functional contribution(s) of this current.. Dog atrial cells were used to investigate the pharmacologic properties of the swelling-induced chloride current. Whole cell patch clamp was used. Swelling-induced chloride current was activated by osmotic stress. Initially, the chloride selectivity and calcium independence of the swelling-induced current in dog atrial cells was demonstrated. Subsequently, a number of putative chloride channel blockers were examined. Anthracene-9-carboxylic acid (1 mM) and dideoxyforskolin (100 microM) and extracellular cAMP (5 mM) were found to partially inhibit the swelling-induced chloride current (approximately 50%, 80%, and 10% inhibition, respectively). Niflumic acid (100 microM), nitrophenylpropylamino benzoate (NPPB; 10 to 40 microM), and (+) 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxy-1H-inden -5-yl)oxy d acetic acid (indanyloxyacetic acid; IAA-94; 100 microM) could fully inhibit the swelling-induced chloride current without decreasing cell size. DIDS (100 microM) and dinitrostilbene disulfonic acid (DNDS; 5 mM) fully inhibited outward currents but only partially inhibited inward current.. Niflumic acid, IAA-94, and NPPB were identified as full blockers of cardiac swelling-induced chloride current. Nonspecific effects were identified for each of the full blockers. Experiments that use these agents as functional antagonists should be carefully designed and interpreted with caution.

Topics: Animals; Anthracenes; Atrial Function; Calcium; Chloride Channels; Colforsin; Dogs; Electrophysiology; Glycolates; Heart Atria; Niflumic Acid; Nitrobenzoates; Patch-Clamp Techniques; Stilbenes

We showed previously that the disulfonic stilbene DNDS (4,4'-dinitrostilben-2,2'-disulfonic acid) was a potent blocker of outwardly rectifying chloride channels (ORCC). The studies reported here were designed to quantify the relationship between electron withdrawal by the 4,4'-substituents and blocker potency. Specifically we compared the blocking effects and molecular properties of the symmetrically substituted 4,4'-diaminostilben-2, 2'-disulfonic acid (DADS) and the hemi-substituted 4-amino, 4'-nitrostilben-2,2'-disulfonic acid (ANDS) with those of DNDS. Blockade was studied using outwardly rectifying colonic chloride channels incorporated into planar lipid bilayers. DADS was 430-fold and ANDS 44-fold less potent than DNDS as blockers of ORCC. Amplitude distribution analysis revealed that all three disulfonic stilbenes act as open channel blockers. Furthermore, this kinetic analysis indicated that the lower potency of DADS and ANDS was due to an increase in off rate. These results support the conclusion that the 4,4'-substituents make an important contribution to blockade by stabilizing the channel-blocker complex. Isopotential electron contour maps illustrated the dramatic shift in charge at the 4,4'-poles of the disulfonic stilbene molecule from electronegative in DNDS to electropositive in DADS as well as the bipolar contour of ANDS. Thus, the greater potency of DNDS results from the symmetric electronegative regions at the 4,4'-poles of the molecule. We hypothesize that the channel protein has two corresponding electropositive areas at the blocker binding site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Calorimetry; Cell Membrane; Chloride Channels; Colon; Dexamethasone; Dose-Response Relationship, Drug; Epithelium; Female; Membrane Potentials; Models, Molecular; Naphthalenesulfonates; Rats; Rats, Sprague-Dawley; Stilbenes

The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4'-dibenzamidostilbene-2,2'-dibenzamidostilbene-2,2'-disu lfonate) and H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) by more than 10-fold at constant ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Regulation; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Chlorides; Erythrocyte Membrane; Flow Injection Analysis; Fluorometry; Humans; Kinetics; Models, Chemical; Stilbenes

To determine the effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on the isotonic volume of jejunal crypt epithelial cells, we isolated these cells by sequential perfusion using a hyperosmolar Ca(2+)-free solution and measured cell volume electronically. 8-BrcAMP caused rapid shrinkage to a reduced but stable cell volume; this isotonic volume reduction was prevented by either a Cl(-)-channel blocker, anthracene-9-carboxylate (A-9C), or Ba2+, a K(+)-channel blocker. 8-BrcAMP substantially increased the rate of A-9C-sensitive 36Cl efflux from crypt cells; this increased rate of efflux was not influenced by Ba2+ but was abolished by alterations in membrane potential. Following 8-BrcAMP-stimulated isotonic volume reduction, addition of either Ba2+ or A-9C caused the crypt cells to reswell. In contrast, 8-BrcAMP added to enterocytes isolated from the villus compartment did not result in A-9C-sensitive volume reduction or in an increased rate of 36Cl efflux. Our data demonstrate that epithelial cells isolated from jejunal crypt compartments respond directly to cAMP with a rapid volume reduction that is paralleled by an increase in 36Cl efflux through a conductive pathway. Unlike other Cl- secretory epithelial cells, the intestinal crypt cell does not appear to regulate its volume in an isotonic medium after cAMP-induced shrinkage.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Anthracenes; Barium; Chlorides; Cyclic AMP; Guinea Pigs; Intestinal Mucosa; Jejunum; Male; Radioisotopes; Stilbenes

Chloride channels were reconstituted into planar lipid bilayers isolated from a preparation of growth cone particles (GCPs) isolated from fetal rat brain. One type of channel was predominantly seen and some of its biophysical and pharmacological properties were studied. The single channel i-V relationship was curvilinear with a chord conductance of 75 pS at +30 mV in symmetric 200 mM NaCl solutions buffered with phosphate. The channel was inactivated by depolarization, and this inactivation was reversed rapidly upon returning to -25 mV. The Cl- channel was significantly permeant to Na+ ions (PNa/PCl = 0.26), and the relative halide permeabilities were determined to be: I(1.92) > Br(1.73) > Cl(1.0) > F(0.34). The channel was inhibited by the common stilbene compounds (DIDS, SITS, DNDS), as well as by Zn2+ ions and an indanyloxyacetic acid derivative. A developmental role for the GCP Cl- channel is suggested by the observation that adult rat brain synaptosomal membranes were nearly devoid of this type of Cl- channel.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Brain; Cell Membrane; Chloride Channels; Membranes, Artificial; Rats; Retinal Cone Photoreceptor Cells; Stilbenes; Synaptosomes

1. We characterized chloride currents in freshly isolated rabbit osteoclasts using whole-cell and single channel patch-clamp recording configurations. Depolarization activated an outwardly rectifying current in 40-50% of cells, distinct from the inwardly rectifying K+ current we have previously reported in osteoclasts. 2. The outwardly rectifying current persisted under conditions where all K+ currents were blocked. Furthermore, the outward current was reversibly inhibited by Cl- transport blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS); 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS); 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS); and niflumic acid. The blocked current had a reversal potential close to the predicted chloride equilibrium potential and was dependent on the chloride concentration gradient. 3. In those osteoclasts in which outwardly rectifying current was not initially apparent, exposure to hyposmotic extracellular solution resulted in its reversible activation. The induced current was due to Cl-, based on its reversal close to the chloride equilibrium potential and sensitivity to blockade by Cl- channel inhibitors. The hyposmotically induced current could be activated in Ca(2+)-free solutions containing 0.2 mM EGTA. 4. When studied in the current-clamp configuration, hyposmotic stimulation caused depolarization from -76 +/- 5 to -5 +/- 6 mV (mean +/- S.D., n = 7). 5. Unitary Cl- currents were recorded in the cell-attached patch configuration at positive potentials. Single channels had a slope conductance of 19 +/- 3 pS (n = 5). Reduction of the external [Cl-] shifted the current-voltage relationship in the positive direction, supporting the conclusion that these were Cl- currents. Like the whole-cell currents, single channel Cl- currents were activated by exposure of cells to hyposmotic bathing solution. 6. We conclude that rabbit osteoclasts express an outwardly rectifying Cl- current that can be activated by osmotic stress. Cl- channels may play a role in cell volume regulation and may also provide conductive pathways for dissipating the potential difference that arises from electrogenic proton transport during bone resorption.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chloride Channels; Chlorides; Electric Conductivity; Electrophysiology; Osmolar Concentration; Osteoclasts; Potassium; Rabbits; Stilbenes; Stimulation, Chemical

Volume expansion of little skate (Raja erinacea) erythrocytes leads to the formation of oligomeric forms of the band 3 protein. The oligomers are formed following incubation in hypotonic media or inclusion of a permeant solute such as ethylene glycol. Oligomers were detected by specifically labeling the skate erythrocyte band 3 homolog with the radiolabeled stilbene [3H]4,4'-diisothiocyano-1,2 diphenylethane-2,2'-disulfonic acid and cross-linking membrane proteins with the homobifunctional agent bis(sulfosuccinimidyl) suberate. Under isoosmotic conditions, the distribution of band 3 in the monomer, dimer, and tetramer forms was 38, 54, and 8%. Medium of 0.5 osmolarity caused the distribution to change to 22% monomer, 31% dimer, and 47% tetramer. Upon return to isoosmotic conditions, cell volume, as well as the distribution of band 3 in the monomer, dimer, and tetrameric forms, returned to control values. Return of hypotonically swollen cells to isoosmotic conditions also returned taurine efflux back to control values. Hyperosmolarity caused a 20% shrinkage of cell volume but did not have any effect on either taurine efflux or the distribution of band 3 in the oligomeric forms. Pyridoxal 5'-phosphate and dinitrostilbene disulfonate, two agents that interact with and cause the formation of the tetrameric form of human band 3, also caused a shift toward to tetrameric peak in skate band 3. To determine whether the cross-linked oligomers had proteins that closely associate with band 3 in the intact cell, Western blots were performed to detect ankyrin and band 4.1. Neither protein appeared with the dimeric or tetrameric forms of band 3, suggesting that the mobility shift was in fact due to association of band 3 monomers. This is the first demonstration that oligomerization of band 3 occurs during a physiologic effect that may be mediated by the band 3 protein.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Ankyrins; Cell Size; Cytoskeletal Proteins; Erythrocytes; Membrane Proteins; Neuropeptides; Osmolar Concentration; Pyridoxal Phosphate; Skates, Fish; Stilbenes

Cl-formate, Cl-oxalate, and SO4-CO3 exchange participate in Cl and organic anion transport across the brush border membrane of the rabbit proximal tubule. To determine the functional similarity of these transporters to each other and to band 3, we characterized, in isolated membrane vesicles, the inhibition of these transporters by compounds known to inhibit erythrocyte band 3. 4,4'-Dinitro-2,2'-disulfonic stilbene (DNDS), diphenylamine-2-carboxylate (DPC), flufenamate, and 4-aceto-4'-isothiocyano-2,2'-disulfonic stilbene (SITS) were effective inhibitors of Cl-oxalate and SO4-CO3 exchange, suggesting at least some common structural motifs between these exchangers and band 3. Cl-formate exchange was relatively insensitive to DNDS and DPC but sensitive to flufenamate (IC50 = 43 microM). Sensitivity to DNDS but not 4-amino-4'-amino-2,2'-disulfonic stilbene, a feature of band 3, was seen only for the SO4-CO3 exchanger. None of the exchangers had significant affinity for dipyridamole, furosemide, or probenecid. Finally, the presence of DPC or flufenamate increased the IC50 for reversible inhibition by DNDS, consistent with at least a partial overlap between the disulfonic stilbene and diphenylamine carboxylate binding sites of the Cl-oxalate exchanger. We next examined the effect of irreversible SITS binding. The Cl-oxalate exchanger was inhibited 90%, consistent with its high affinity for reversible inhibition by stilbenes. SITS pretreatment caused 50% inhibition of the Cl-formate exchanger, consistent with the reduced affinity of this exchanger for reversible binding of stilbenes. Despite the high sensitivity of the SO4-CO3 exchanger to reversible inhibition by stilbenes, SITS pretreatment caused < 20% irreversible inhibition of this exchanger. Finally, we characterized the stilbene inhibition of the Cl-oxalate exchanger in more detail. The presence of oxalate increased the IC50 for reversible inhibition by DNDS or SITS, implying that oxalate can directly compete at the reversible stilbene binding site of the Cl-oxalate exchanger. However, oxalate could not protect against covalent inactivation of the Cl-oxalate exchanger by SITS, indicating the presence of a separate site for irreversible binding of disulfonic stilbenes. These results suggest a dissociation between the sensitivities of proximal tubule anion exchangers to reversible and irreversible inhibition by disulfonic stilbenes. In contrast to band 3, the Cl-oxalate exchanger must possess separate site

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Antiporters; Biological Transport; Carbonates; Chlorides; Dose-Response Relationship, Drug; Flufenamic Acid; Kidney Cortex; Male; Microvilli; Oxalates; Rabbits; Stilbenes; Sulfates

Ion channels in endosomal membranes from rabbit kidney cortex were studied after reconstitution into planar lipid bilayers. The most frequently observed ion channel was anion selective (PCl/PK = 13) and had a single-channel conductance of 116 pS when the cis and trans solutions contained 410 and 150 mM KCl, respectively, and a conductance of 90 pS in symmetrical 150 mM KCl solutions. The anion selectivity sequence of the channel was NO3- > F- > Br- > Cl- >> I-. The activity of the channel was voltage dependent such that hyperpolarization of the cis, or cytoplasmic, surface of the channel increased the open probability (Po). The activity of the channel was also highly dependent on the calcium activity of the cis but not the trans solution. Channels were fully active (Po > 0.7) at Ca2+ concentration > 1 microM, but channel activity was completely absent (Po < 0.001) at Ca2+ concentration < 250 nM. The effects of calcium on Po were not voltage dependent. The Cl(-)-channel blocker 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5- yl)oxy]-acetic acid (IAA-94/95) produced a concentration-dependent reversible flickering block of the endosomal channel with a Ki of 15 microM. 4,4'-Dinitrostilbene-2,2'-disulfonic acid, a disulfonic stilbene, also produced a flickering block of the channel with a Ki of approximately 5 microM. Because endosomal Cl- channels are believed to facilitate endosomal acidification, we tested the effects of IAA-94/95 and deletion of Ca2+ on the rate of acidification of intact endosomes. Because neither maneuver affected acidification, we conclude that the 116-pS channel does not participate in endosomal acidification. This channel may be involved in other endosomal processes, e.g., cell volume regulation and control of membrane trafficking.

Topics: Acids; Adenosine Triphosphate; Animals; Calcium; Chloride Channels; Cyclic AMP-Dependent Protein Kinases; Female; Glycolates; Ion Channel Gating; Kidney Cortex; Organelles; Rabbits; Stilbenes

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.

Topics: Anion Exchange Protein 1, Erythrocyte; Benzenesulfonates; Chlorides; Eosine Yellowish-(YS); Erythrocyte Membrane; Erythrocytes; Erythrosine; Humans; Kinetics; Mathematics; Models, Biological; Nystatin; Protein Conformation; Stilbenes

Effects of stilbene disulfonates on single KATP 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-acetamido-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 KATP 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. KATP channel inhibition was concentration dependent with an order of potency of DIDS > SITS approximately 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 (Po) 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 KATP channel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation i

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Cell Membrane; Guinea Pigs; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Fluid; Kinetics; Membrane Potentials; Myocardium; Potassium Channel Blockers; Potassium Channels; Stilbenes

Transepithelial and cell membrane potential measurements have suggested that the basolateral membrane of gerbil vestibular dark cells contains Cl- conductive pathways. We used the patch clamp technique to search this membrane for Cl- conductive channels which could account for the macroscopic observations. Two types of Cl- channel were found in both cell-attached and excised membrane patches. One type was found with an incidence of 19% and had a single-channel conductance of 95 +/- 1 pS (N = 20) in symmetrical Cl- solutions. The other type was found with an incidence of 3% and had a large single-channel conductance of 360 +/- 11 pS (N = 12) in symmetrical Cl- solutions (LC-type Cl- channel). Both types of Cl- channel had linear current-voltage relations and at least 2 substates. In asymmetrical Cl- solutions (gluconate substitution) the current-voltage relations fit the Goldman-Hodgkin-Katz current equation for Cl-. Neither channel was blocked by Zn2+, NPPB, DIDS, DNDS or quinine. The 95 pS channel exhibited a spontaneous 'rundown' of its activity within 1 to 10 min after being excised. This rundown was not reversed by the catalytic subunit of protein kinase A. Channel activity was not dependent on the presence of cytosolic Ca2+ nor markedly altered by variations in cytosolic pH between 6.5 and 8.0. The two Cl- channels were distinguished by the membrane voltage ranges in which they were active and by their anion selectivity. The open probability of the 95 pS channel was insensitive to voltage and the anions NO3-, I- and Br- were only half as permeable as Cl-. By contrast, the LC-type Cl- channel was mostly active between about +/- 30 mV and equally permeable to NO3-, I-, Br- and Cl-. The 95 pS Cl- channel may account for the observed transepithelial and intracellular voltage responses to Cl- concentration steps and provide the path for the recirculation of Cl- across the basolateral membrane. The LC-type Cl- channel shows the same lack of anion discrimination as the anion pathway activated during hyposmotic challenge.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chloride Channels; Chlorides; Electric Conductivity; Electrophysiology; Gerbillinae; Membrane Potentials; Nitrobenzoates; Quinine; Stilbenes; Vestibule, Labyrinth; Zinc

One important mechanism for reabsorption of chloride in the kidney proximal tubule involves anion exchange of chloride for a base. Anion exchange transport systems in general demonstrate sensitivity to inhibition by disulfonic stilbenes, probenecid, furosemide, and the arginyl amino group modifier phenylglyoxal. Using disulfonic stilbene affinity chromatography, we have identified and partially purified a protein with anion exchanger properties in luminal membrane vesicles isolated from rabbit kidney cortex. This protein has a molecular weight of 162 kD. The binding of the 162 kD protein to the stilbene affinity matrix is inhibited by disulfonic stilbenes, probenecid, furosemide, and phenylglyoxal. Reconstitution of the proteins eluted from the affinity matrix into liposomes demonstrates anion exchange activity as assayed by radiolabeled chloride influx. Deletion of the 162 kD protein from the eluted mixture by probenecid diminishes the anion exchanger activity in the reconstituted liposomes. Further purification of the disulfonic stilbene column eluant by Econo-Pac Q ion exchange chromatography resulted in significant enrichment in 162 kD protein abundance and also anion exchange activity in reconstituted liposomes. The results of the above experiments strongly suggest that the 162 kD protein is an anion exchanger. Insight into the functional and molecular characteristics of this protein should provide important information about the mechanism(s) of chloride reabsorption in the kidney proximal tubule.

Topics: Animals; Anion Transport Proteins; Carrier Proteins; Chlorides; Furosemide; In Vitro Techniques; Ion Transport; Kidney Tubules, Proximal; Microvilli; Molecular Weight; Probenecid; Proteolipids; Rabbits; Stilbenes

Brush-border membrane (BBM) proteins that bind the arginine-specific reagent phenylglyoxal (PG) and interact with stilbene disulfonic derivatives were identified in canine kidney cortex. Pretreatment of BBM vesicles with PG resulted in irreversible inhibition of anion exchange as assayed by 36Cl- influx mediated via Cl-/Cl- exchange. Cl-/Cl- exchange was reversibly inhibited by the disulfonic stilbene 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). A stilbene-affinity matrix was prepared by immobilizing DNDS in polyacrylamide beads. Elution of the BBM proteins from a disulfonic stilbene (DNDS) affinity matrix revealed two proteins at 160 and 230 kDa that were significantly enriched compared to initial material. Radiolabeling of the eluted mixture with [14C]phenylglyoxal demonstrated covalent binding to several proteins, including the 160 kDa protein. Reconstitution of the proteins eluted from the affinity matrix into phosphatidylcholine demonstrated DIDS-sensitive 36Cl(-)-influx mediated via Cl-/Cl- exchange. Pretreatment of the BBM vesicles with PG selectively blocked binding of the 160 kDa protein to the DNDS affinity matrix. Radiolabelling of the PG-pretreated, affinity-purified membrane proteins showed selective prevention of [14C]phenylglyoxal binding to the 160 kDa protein. Reconstitution of the PG-pretreated proteins eluted from the affinity matrix demonstrated significant reduction in Cl-/Cl- exchange activity. These results suggest that a 160 kDa protein is a strong candidate for anion exchange transport in kidney proximal tubules.

Topics: Animals; Anions; Chlorides; Dogs; Furosemide; Kidney Cortex; Membrane Proteins; Microvilli; Phenylglyoxal; Stilbenes

Lung carbonic anhydrase (CA) permits rapid pH responses when changes in regional ventilation or perfusion alter airway and alveolar PCO2. These pH changes affect airway and vascular resistances and lung compliance to optimize the balance of regional ventilation (VA) and perfusion (Q) in the lung. To test the hypothesis that these or other CA-dependent mechanisms contribute to VA/Q matching, we administered acetazolamide (25 mg/kg intravenously) to six anesthetized and paralyzed dogs and measured VA/Q relationships before and after CA inhibition by the multiple inert gas elimination technique. Four other groups of dogs were studied to control for possible confounding effects of time under anesthesia and nonselective CA inhibition by acetazolamide: (a) saline placebo as a control for duration of anesthesia, (b) 4% CO2 inhalation to mimic systemic CO2 retention, (c) 1 mg/kg benzolamide (a selective renal CA inhibitor) or 0.5 meq/kg HCl to mimic systemic metabolic acidosis, and (d) 500 mg/kg 4,4'-dinitrostilbene-2,2'-disulfonate (an inhibitor of red cell band 3 protein) to mimic the respiratory acidosis arising from an intracapillary block to rapid mobilization of plasma HCO3- in CO2 exchange. Acetazolamide increased VA/Q mismatch and reduced arterial PO2 measured at equilibrium but these did not occur in the control group. There was no deterioration in VA/Q matching when systemic respiratory acidosis produced either by CO2 inhalation or 4,4'-dinitrostilbene-2,2'-disulfonate or metabolic acidosis (benzolamide or HCl) were imposed to mimic the effects of acetazolamide apart from its inhibition of lung CA. These results support the concept that lung CA subserves VA/Q matching in the normal lung.

Topics: Acetazolamide; Acidosis; Acidosis, Respiratory; Animals; Benzolamide; Blood Pressure; Carbon Dioxide; Carbonic Anhydrases; Cardiac Output; Dogs; Heart Rate; Hemodynamics; Lung; Oxygen; Partial Pressure; Pulmonary Circulation; Respiration; Stilbenes

Stilbenedisulfonic acid derivatives have been shown to block Cl- channels directly in many different preparations. Therefore, the utility of these compounds as tools for studying the cAMP-dependent Cl- current (ICl) in guinea-pig ventricular myocytes was examined using the patch-clamp technique to record whole-cell Cl- currents at room temperature. It was found that 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) increased, rather than decreased, the isoproterenol (ISO)-activated Cl- current. However, SITS alone stimulated little or no sustained current, suggesting that SITS activates the Cl- current through a synergistic effect with ISO. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) also enhanced the ISO-activated Cl- current. However, 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) did not have any effect. SITS also exhibited a synergistic effect on the ISO-enhanced Ca2+ current in the same cells, suggesting that it affects the pathway involved in beta-adrenergic regulation of both Cl- and Ca2+ channels. SITS had no effect on the Cl- current stimulated by direct activation of adenylate cyclase with forskolin or exposure to the membrane-permeable cAMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate. This suggests that SITS and DIDS may enhance the ISO-activated Cl- current via an effect on the beta-adrenergic receptor. It is concluded that these stilbenedisulfonic acid derivatives are not effective antagonists of cAMP-activated Cl- channels in cardiac ventricular myocytes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium Channels; Chloride Channels; Cyclic AMP; Guinea Pigs; Heart; In Vitro Techniques; Isoproterenol; Membrane Potentials; Membrane Proteins; Myocardium; Stilbenes

Irreversible inhibition, 99.8% of control values for chloride transport in human red blood cells, was obtained by well-established methods of maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The kinetics of the residual chloride transport (0.2%, 106 pmol.cm-2 x s-1) at 38 degrees C, pH 7.2) was studied by means of 36Cl- efflux. The outside apparent affinity, expressed by Ko1/2,c, was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Borders, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemistry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of the residual chloride flux depended on the extracellular chloride concentration, but was not purely competitive. The half-inhibition concentrations at [Cl(o)] = 150 mM in control cells (Ki,o) and covalently DIDS-treated cells (Ki,c) were: DIDS, Ki,c = 73 nM; DNDS, Ki,o = 6.3 microM, Ki,c = 22 microM; phloretin, Ki,o = 19 microM, Ki,c = 17 microM; salicylate, Ki,o = 4 mM, Ki,c = 8 mM; Killer III, Ki,o = 10 microM, Ki,c = 10 microM.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetophenones; Chloride Channels; Dinitrophenols; Erythrocytes; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Membrane Proteins; Phloretin; Salicylates; Salicylic Acid; Stilbenes; Temperature

Cardiac sarcoplasmic reticulum (SR) has several chloride (Cl-) channels, which may neutralize the charge across the SR membrane generated by Ca2+ movement. We recently reported a novel 116-picosiemen Cl- channel that is activated by protein kinase A-dependent phosphorylation in cardiac SR. This Cl- channel may serve as a target protein in the receptor-dependent regulation of cardiac excitation-contraction coupling. To understand further regulatory mechanisms, the effects of Ca2+ on the Cl- channel were studied using the planar lipid bilayer-vesicle fusion technique. In the presence of calmodulin (CaM, 0.1 mumol/L per microgram SR vesicles), Ca2+ (3 mumol/L to 1 mmol/L) added to the cis solution reduced the channel openings in a concentration-dependent fashion, whereas Ca2+ (1 nmol/L to 1 mmol/L) alone or CaM (0.1 to 1 mumol/L per microgram SR vesicles) with 1 nmol/L Ca2+ did not affect the channel activity. This inhibitory effect of Ca2+ in the presence of CaM was prevented by CaM inhibitors N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide and calmidazolium but not by CaM kinase II inhibitor KN62. These results suggest that the Ca(2+)-CaM complex itself, but not CaM kinase II, is involved in this channel inhibition. Thus, the cardiac SR 116-picosiemen Cl- channel is regulated not only by protein kinase A-dependent phosphorylation but also by the cytosolic Ca(2+)-CaM complex. This is a novel second messenger-mediated regulation of Cl- channels in cardiac SR membrane.

Topics: Animals; Calcium; Calmodulin; Chloride Channels; In Vitro Techniques; Membrane Proteins; Myocardium; Protein Kinases; Sarcoplasmic Reticulum; Stilbenes; Swine

Near-UV circular dichroism (CD) was used to identify differences in the tertiary structure of human erythrocyte band 3, the chloride/bicarbonate exchange protein, consequent to covalent binding of anion transport inhibitors to the intramonomeric stilbenedisulfonate (ISD) site. Isolated intact band 3 and its membrane domain (B3MD) were compared. Spectral differences were observed which involved intradomain effects, in that they were seen both with intact band 3 and with B3MD, or interdomain effects, in that they were observed only for B3MD, but were inhibited when the cytoplasmic domain was attached. The intradomain effect involved a significant loss in optical activity in the Phe/Tyr region of the spectrum below 280 nm. It was seen only when the ISD site had stilbenedisulfonates bound covalently at pH 7.4. Raising the pH to 9.6 after adduct formation "normalized" this spectral change irreversibly. The interdomain effect was identified in the Trp spectral region at 292 nm. There was a significant increase in optical activity at 292 nm when bulky covalent ligands such as DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) were bound to B3MD, but not when the same ligands were bound to intact band 3. These latter results offer evidence that certain aspects of the conformational response of the integral domain are inhibited by the presence of an attached cytoplasmic domain. The potential significance of interdomain interactions to band 3 function is discussed briefly.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Circular Dichroism; Humans; Protein Structure, Tertiary; Spectrophotometry, Ultraviolet; Stilbenes

The contributions to pulmonary gas exchange of red blood cell (RBC) membrane band 3 protein HCO3(-)-Cl- exchange and carbonic anhydrase- (CA) catalyzed HCO3- dehydration have never been determined directly in the whole animal. We utilized an experimental and model approach to measure these by analysis of phase III exhaled CO2 and O2 profiles in anesthetized dogs. In this method, we inhibit RBC membrane band 3 protein and cytoplasmic CA in RBCs passing the pulmonary capillaries and lung vascular luminal membrane-bound CA during a single ventilatory cycle. This is achieved with appropriately timed right atrial infusions of 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to inhibit band 3 protein, ethoxzolamide (a lipophilic CA inhibitor with rapid membrane penetrance) to inhibit RBC and lung tissue CA, and benzolamide (an extremely hydrophilic CA inhibitor with virtually no penetrance into RBC cytoplasm) to inhibit only lung vascular luminal membrane CA. DNDS caused a 15% reduction in CO2 production (VCO2) without any change in O2 consumption (VO2). The addition of benzolamide to DNDS did not cause any further decrease in VCO2. Inhibition of RBC CA by ethoxzolamide caused a 67% reduction in VCO2 and a 11.5% reduction in VO2. Inhibition of lung vascular CA by benzolamide alone caused no statistically significant changes in either VCO2 or VO2. These results are in general agreement with in vitro data and model calculations. The only exceptions are the higher than predicted effect of RBC CA inhibition on VO2 (Bohr effect) and the lack of any contribution to CO2 transfer in the dog by lung vascular CA with access to plasma as a possible consequence of an endogenous plasma CA inhibitor.

Topics: Acetylene; Animals; Anion Exchange Protein 1, Erythrocyte; Benzolamide; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Dogs; Erythrocytes; Ethoxzolamide; Hematocrit; Lung; Mass Spectrometry; Oxygen Consumption; Pulmonary Gas Exchange; Respiration, Artificial; Stilbenes

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl- channel (ORDIC) during cAMP-mediated Cl- secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl- channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4'-dinitro-2,2'-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ODIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl- secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC50 = 0.5 +/- 0.3 mumol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl- secretion unless high concentrations were used (IC50 = 240 +/- 10 mumol/l). In order to exclude effects of NPPB other than on the apical Cl- channel, transepithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl- gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl- current showed a very-low-frequency Lorentzian noise component (fc = 1.4 +/- 0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Calcium; Cell Membrane; Chlorides; Colforsin; Cyclic AMP; Electrophysiology; Fura-2; Humans; Intestinal Mucosa; Intracellular Membranes; Ion Channels; Nitrobenzoates; Stilbenes; Tumor Cells, Cultured

1. Stretch-activated anion currents were studied in sino-atrial and atrial cells using the whole-cell patch clamp technique. With continuous application of positive pressure (5-15 cmH2O) through the patch clamp electrode, the cell was inflated and the membrane conductance was increased. 2. Voltage clamp steps revealed that the stretch-activated currents had time-independent characteristics. The increase in membrane conductance was reversible on subsequent application of negative pressure to the electrode. 3. The reversal potential of the stretch-activated currents was shifted by 60 mV for a 10-fold change in intracellular Cl- concentration, while it was unaffected by replacement of Na+ in the extracellular solution by N-methyl-D-glucamine. Cell superfusion with Cl(-)-deficient solution (10 mM Cl-) reduced the amplitude of outward current. These findings indicate that the stretch-activated conductance is Cl- selective. 4. The sequence of anion permeability through the stretch-activated conductance was determined to be I-(1.7) > NO3-(1.5) > Br-(1.2) > Cl-(1.0) > and F-(0.6). SCN- appeared to be more permeant than I-. 5. The stretch-activated conductance was reduced by the Cl- channel blockers, 4,4'-dinitrostilbene-2,2'-disulphonic acid disodium salt, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid or anthracene-9-carboxylate (9-AC). Administration of furosemide or bumetanide had no effect. 6. The stretch-activated Cl- current was recorded even though intracellular Ca2+ ions were chelated by including 10 mM EGTA in the pipette solution. Neither the specific peptide inhibitor of cyclic AMP-dependent protein kinase (50 microM), nor the non-selective blocker of protein kinases, H-7 (20 microM), was effective in reducing the stretch-activated Cl- current, suggesting that the stretch-activated Cl- current is a novel type of cardiac Cl- current, which shows a different modulatory mechanism from that of other cardiac Cl- currents.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Chlorides; In Vitro Techniques; Ion Channels; Membrane Potentials; Myocardial Contraction; Myocardium; Potassium Channels; Rabbits; Sinoatrial Node; Sodium; Stilbenes; Time Factors

Prompted by reports of sexual impotence among chemical factory workers exposed to amsonic acid (4,4'-diaminostilbene-2,2'-disulfonic acid; CAS 81-11-8) and its synthetic precursors 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNSDSA; CAS 128-42-7), 2-methyl-5-nitrobenzenesulfonic acid (MNBSA; CAS 121-03-09), and 4-nitrotoluene (CAS 99-99-0), the uterine-weight-increasing actions of single intraperitoneal doses of these chemicals were determined at 24 h after treatment in weanling female rats and compared to the results of similar experiments with diethylstilbestrol (DES; CAS 56-53-01), a synthetic estrogen chemically related to amsonic acid and DNSDSA. Doses of 100 mg/kg or less of amsonic acid were either without effects or produced equivocal effects, while uterine weights were increased after doses of 300 and 1000 mg/kg; doses of 3000 mg/kg were clearly toxic. Neither DNSDSA nor MNBSA increased uterine weight after doses that were not overtly toxic. Doses of 10 mg/kg or less of 4-nitrotoluene were without effect, but doses of 30 and 100 mg/kg increased uterine weights without producing overt toxicity; doses of 1000 mg/kg were clearly toxic. While both amsonic acid and 4-nitrotoluene exhibited uterotropic effects, they were both much weaker than DES in this respect. Other experiments indicated that the time course of the effects of approximately equiactive doses of amsonic acid and DES were very similar, and that the responses to oral doses of amsonic acid were not appreciably different from the responses to the same doses given intraperitoneally. Finally, a sample of amsonic acid taken from the workplace of the complaining workers was also found to have uterotropic activity. These experiments suggest that amsonic acid and 4-nitrotoluene have estrogenic activity, and thus provide a possible mechanistic explanation for the complaints of impotency in factory workers exposed to these substances.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Benzenesulfonates; Corn Oil; Diethylstilbestrol; Estrogens, Conjugated (USP); Female; Occupational Exposure; Organ Size; Rats; Rats, Inbred Strains; Sodium Chloride; Stilbenes; Time Factors; Toluene; Uterus

Chloride channels at the apical membrane of intestinal epithelial cells are involved in the excessive fluid secretion in diarrhea and diminished secretion in cystic fibrosis (CF). Diarrhea induced by heat-stable toxin from Escherichia coli is associated with elevated guanosine 3',5'-cyclic monophosphate (cGMP) in intestinal epithelial cells, but it is unknown whether chloride secretion is regulated by cGMP directly or via cGMP-dependent protein kinase (PKG). Single-channel recordings (inside-out excised patches) from the apical membrane of T84 cells reveal a 10-pS chloride channel with a linear current-voltage relationship, which is opened when an endogenous membrane-bound PKG is activated with ATP (1 mM) and cGMP (100 microM). Soluble PKG (200 nM) isolated from bovine lung, added to the intracellular face of patches, also opens this channel. No activation occurs with Ringer solution alone or only ATP or cGMP. Addition of nonhydrolyzable forms of ATP (AMP-PNP, 1 mM) or a combination of ATP, cGMP, plus H-8 (5 microM), an inhibitor of PKG, also does not stimulate the channel. The catalytic subunit of adenosine 3',5'-cyclic mono-phosphate-dependent protein kinase (PKA, 200 nM, with 1 mM ATP) activates a channel with similar characteristics. The 10 pS channel has a PNa/PCl ratio of 0.06, an anion selectivity of Br- (1.2) greater than Cl- (1.0) greater than I- (0.8) greater than F- (0.4), and a low affinity for the chloride channel blockers, 4,4-dinitrostilbene-2,2-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Carcinoma; Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic GMP; Electric Conductivity; Humans; Ion Channel Gating; Membrane Proteins; Nitrobenzoates; Protein Kinases; Stilbenes; Tumor Cells, Cultured

Possible arrhythmogenic effects of the isoproterenol-activated Cl- current were examined in isolated guinea-pig ventricular myocytes under various intra- and extracellular Cl- concentrations. Experiments were carried out with external K+ concentration ([K+]o) decreased to 2 or 3 mM. Under symmetrical concentrations of Cl- in intra- and extra-cellular solutions (ECl = 0 mV), 1 microM isoproterenol (ISP) depolarized resting membrane potential (RMP) by 6.2 +/- 1.1 mV and slowed repolarization with induction of early afterdepolarizations (EADs) in 9 out of 9 cells. EADs appeared at voltages positive to -40 mV, where L-type Ca2+ current is assumed to be activated. When Cl- concentrations were settled near physiological conditions (ECl = -40 - -50 mV), ISP depolarized RMP by 2.8 +/- 0.4 mV and elicited abnormal repolarization with occasional EADs in 6 out of 19 cells. When ECl was set to -80 mV, however, ISP depolarized RMP by only 0.5 +/- 0.5 mV without induction of abnormal activities. Thus, depolarizing effects of ISP and incidence of repolarization abnormalities including EADs were increased as ECl shifted to more positive potential levels. At [K+]o = 4 mM, no abnormal activities were observed when ECl was around -50 mV (0/8), and 6 out of 6 cells showed abnormal activities when ECl was set to 0 mV. ISP-elicited abnormal activities were abolished by 1 mM DNDS (4,4'-dinitrostilbene-2,2'-disulphonic acid), a blocker for Cl- channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Chloride Channels; Electrophysiology; Guinea Pigs; Heart; In Vitro Techniques; Isoproterenol; Membrane Potentials; Membrane Proteins; Myocardium; Stilbenes

1. Inhibition of 36Cl- efflux in frog muscle by the stilbene disulphonates (SD) SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonate) and DNDS (4,4'-dinitro-stilbene-2,2'-disulphonate) depends on the external pH, while the blocking of Cl- conductance with SITS is independent of pH. The 36Cl- efflux inhibition with DNDS has been studied in thin depolarized muscle fibre bundles in order to examine whether this difference is consistent with one transporter or reflects the existence of two mutually independent conductive and non-conductive SD-sensitive Cl- pathways. 2. The 36Cl- efflux response to a sudden inward KCl gradient was studied. At high pH the efflux decreased as predicted for dominant conductive Cl- single-filing, and at low pH the efflux increased in agreement with dominant non-conductive Cl- antiport. DNDS inhibition resulted in the same response, an efflux decrease, at both high and low pH, suggesting a selective reduction of the non-conductive contribution at low pH. 3. The inhibition of 36Cl- efflux as a function of the DNDS concentration at an external pH of 6.9 showed complex kinetics, which could be described as the sum of two Michaelis-Menten functions with different half-inhibition concentrations of DNDS. 4. The results support the hypothesis that the stilbene disulphonate-sensitive Cl- transport is mediated by two separate transporters rather than one.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Biological Transport, Active; Chlorides; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Muscles; Potassium Chloride; Rana temporaria; Stilbenes

The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocyanatostilbenes H2DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC50) of KCl cotransport was 60 microM in the absence of external K and 3 microM at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4' isothiocyanatostilbene-2,2'-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mM. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Calcimycin; Carrier Proteins; Chlorides; Dose-Response Relationship, Drug; Erythrocyte Deformability; Erythrocytes; Ethylmaleimide; K Cl- Cotransporters; Potassium; Sheep; Stilbenes; Symporters

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amino Acid Sequence; Animals; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Biophysical Phenomena; Biophysics; Female; Kinetics; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Oocytes; Stilbenes; Xenopus

The effect of mutation of either Lys 558 or Lys 869 or both on mouse erythroid band 3 protein (AE1)-mediated 36Cl- efflux and its inhibition by pyridoxal 5-phosphate (P5-P), DNDS and H2DIDS were studied. Regardless of the mutation, band 3 was always capable of executing Cl- self-exchange. P5-P (5 mM, pH 7.6) produced irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues were replaced, mutant (NM), irreversible inhibition could no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues, 558 or 869. Inhibition by DNDS changed dramatically upon mutation. The Ki app increased from 6.0 microM in the wild type (KK) to 23 microM in the mutant NK, to 73 microM in the mutant KM and to 474 microM in the double mutant NM. The Km value for activation of the transport system by varying the substrate concentration by isosmotic substitution of Cl- with SO4(2-) decreased from 42 mM in the wild type (KK) to 11.3 mM in the mutant NM. The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate Cl-. In the double mutant NM, H2DIDS is no longer able to produce irreversible inhibition at pH 7.6. This is evidently related to the replacement of Lys 558 (pK 8.2) by Asn 558 in this mutant (see Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., Passow, H., 1989. EMBO J. 8:3601-3609). However, at pH 9.5, some irreversible inhibition could still be observed. This suggests that the other lysine residue (pK 10.8) that is known to be involved in covalent binding with the second isothiocyanate group of H2DIDS is still present, and hence, not identical to Lys 869, which had been substituted by a methionine residue. However, this result remains inconclusive since after mutagenesis, the H2DIDS may produce inhibition at a site that is not normally involved in H2DIDS binding.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amino Acid Sequence; Animals; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Biological Transport; Chlorides; Lysine; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Oocytes; Protein Binding; Pyridoxal Phosphate; Stilbenes; Xenopus laevis

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

Topics: Chloride Channels; Cystic Fibrosis; Electric Conductivity; Electrophysiology; Epithelium; Humans; Membrane Proteins; Reference Values; Stilbenes; Sweat Glands

An electrogenic Na+/HCO3- cotransport system was identified and characterized in freshly dissociated salamander Müller (glial) cells. Under voltage-clamp, these cells generated an outward current when external HCO3- concentration [( HCO3-]o) was raised. This current was Na(+)-dependent, Cl(-)-independent, and was blocked by the stilbenes 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), and by harmaline, demonstrating that the current was generated by a Na+/HCO3- cotransport system. Substantially larger currents were evoked when [HCO3-]o was raised at the Müller cell endfoot as compared to other cell regions, indicating that cotransporter sites are localized preferentially to the endfoot. The reversal potential of the current, which varied as a function of HCO3- and Na+ transmembrane gradients, indicated that the cotransporter has a HCO3-:Na+ stoichiometry of 3:1.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Ambystoma; Animals; Bicarbonates; Electrophysiology; Harmaline; Hydrogen-Ion Concentration; In Vitro Techniques; Neuroglia; Retina; Sodium; Sodium Bicarbonate; Stilbenes

Stilbene-sensitive glycine transport was investigated in human red blood cells and ghosts. We have found that this component of glycine transport was inhibited by the stilbene derivatives 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS); the apparent constant for inhibition by DNDS was 4 microM in the presence of 150 mM chloride. DNDS-sensitive glycine influx was modulated by pH such that as pH was increased from 5.9 to 9.2, transport increased from 2.5 to 140 mumol.kg Hb-1.h-1 at 37 degrees C and 100 microM glycine. The increased transport was correlated with an increase in the amount of glycine present as the anion over this pH range (0.03-40 microM glycine anion), but, in addition, pH had a direct effect on transport. Glycine influx was studied as a function of glycine anion concentration with anion varied by changing pH at a constant total glycine concentration and by changing total glycine at a constant pH. A comparison of these data demonstrated that the stilbene-sensitive glycine anion flux is stimulated by protons with half-maximal stimulation below pH 6.5 and suggests that the glycine anion and a proton are cotransported. Inorganic anions transported by band 3, including Cl, NO3, and SO4, inhibited glycine transport. Glycine flux into resealed ghosts was inhibited by Cl with an inhibition constant of 25 mM. The similarities between the kinetic constants for transport inhibition by Cl and DNDS and the kinetic constants for Cl and DNDS binding to band 3 suggest that the DNDS-sensitive glycine anion and proton cotransport is via band 3.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Transport Proteins; Anions; Biological Transport; Carrier Proteins; Chlorides; Erythrocyte Membrane; Erythrocytes; Glycine; Humans; Hydrogen-Ion Concentration; Osmolar Concentration; Protons; Sodium; Stilbenes

The electrical properties of the apical membrane of isolated rabbit parietal cells were studied using the patch-clamp technique. The apical membrane of the parietal cells plated on Matrigel and maintained in culture conditions was identified by lectin-binding studies. Cell-attached and excised inside-out patches from 10(-4) M cimetidine-treated parietal cells infrequently contained Cl- channels (9% of the patches). A single class of voltage-dependent outwardly rectifying Cl- channels with 24 +/- 1-pS conductance was observed in 75% of the patches from cells stimulated (acid secreting) by 10(-4) M histamine. Other anions passed through these channels with a permeability sequence of I- (1.2) greater than Br- (1.1) greater than or equal to Cl- (1.0) greater than NO3- (0.7) greater than SO4(2-) (0.1), but there was a very low permeability for Na+ or K+ (PCl-/PNa+ or PCl-/PK+ greater than 5). In inside-out patch configurations the Cl- channel was insensitive to Ba2+ and stilbene derivatives but was inhibited by diphenylamine-2-carboxylic acid in a manner characteristic of a reversible open-channel blocker. It is concluded that H2-receptor agonist stimulation of acid secretion by rabbit parietal cells activates Cl- channels in the apical cell membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Aminopyrine; Animals; Cells, Cultured; Chloride Channels; Chlorides; Cimetidine; Gastric Acid; Histamine; Ion Channels; Kinetics; Male; Membrane Potentials; Membrane Proteins; Microscopy, Electron; Microvilli; Parietal Cells, Gastric; Rabbits; Stilbenes

We compared the potency and inhibitory actions of three different classes of organic acids on a Cl channel derived from colonic enterocyte plasma membrane vesicles. Chloride channels were incorporated into planar lipid bilayer membranes to examine the effects of the anthranilic acids, diphenylamine 2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), the indanyl alkanoic acids, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5-yl)oxy] acetic acid (IAA-94) and its stereoenantiomer IAA-95, and the disulfonic stilbene, 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS). Except for DNDS, each of the blockers was equipotent from both the outer membrane and the cytoplasmic side of the channel protein. The potency order from the outmembrane side was DNDS greater than IAA-94 = IAA-95 greater than NPPB much greater than DPC. In contrast, the potency order from the cytoplasmic side was IAA-94 = IAA-95 greater than NPPB greater than DNDS much greater than DPC. DPC and NPPB caused a concentration-dependent decrease in the single-channel conductance (fast block). DNDS, IAA-94, and IAA-95 caused a flickery-type block and a concentration-dependent decrease in open-channel probability. Kinetic analysis revealed that blockade could be explained by a linear closed-opened-blocked kinetic scheme. Similarities in the electrostatic potential maps of these open-channel blockers suggest they may bind to a single shared binding site within the channel protein.

Topics: Animals; Chloride Channels; Chlorides; Colon; Diuretics; Electrophysiology; Female; Glycolates; Ion Channels; Kinetics; Lipid Bilayers; Membrane Proteins; Models, Molecular; Nitrobenzoates; ortho-Aminobenzoates; Rats; Rats, Inbred Strains; Stereoisomerism; Stilbenes

An electrogenic Na+/HCO3- cotransport system was studied in freshly dissociated Müller cells of the salamander retina. Cotransporter currents were recorded from isolated cells using the whole-cell, voltage-clamp technique following the block of K+ conductance with external Ba2+ and internal Cs+. At constant pHo, an outward current was evoked when extracellular HCO3- concentration was raised by pressure ejecting a HCO3(-)-buffered solution onto the surface of cells bathed in nominally HCO3(-)-free solution. The HCO3(-)-evoked outward current was reduced to 4.4% of control by 0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate), to 28.8% of control by 2 mM DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), and to 28.4% of control by 2 mM harmaline. Substitution of choline for Na+ in bath and ejection solutions reduced the response to 1.3% of control. Bicarbonate-evoked currents of normal magnitude were recorded when methane sulfonate was substituted for Cl- in bath, ejection, and intracellular solutions. Similarly, an outward current was evoked when extracellular Na+ concentration was raised in the presence of HCO3-. The Na(+)-evoked response was reduced to 16.2% of control by 2 mM DNDS and was abolished by removal of HCO3- from bath and ejection solutions. Taken together, these results (block by stilbenes and harmaline, HCO3- and Na+ dependence, Cl- independence) indicate that salamander Müller cells possess an electrogenic Na+/HCO3- cotransport system. Na+/HCO3- cotransporter sites were localized primarily at the endfoot region of Müller cells. Ejection of HCO3- onto the endfoot evoked outward currents 10 times larger than currents evoked by ejections onto the opposite (distal) end of the cell. The reversal potential of the cotransporter was determined by DNDS block of cotransport current. In the absence of a transmembrane HCO3- gradient, the reversal potential varied systematically as a function of the transmembrane Na+ gradient. The reversal potential was -0.1 mV for a [Na+]o:[Na+]i ratio of 1:1 and -25.2 mV for a Na+ gradient ratio of 7.4:1. Based on these values, the estimated stoichiometry of the cotransporter was 2.80 +/- 0.13:1 (HCO3-:Na+). Possible functions of the glial cell Na+/HCO3- cotransporter, including the regulation of CO2 in the retina and the regulation of cerebral blood flow, are discussed.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Ambystoma; Animals; Bicarbonates; Carbon Dioxide; Carrier Proteins; Harmaline; Hydrogen-Ion Concentration; Osmolar Concentration; Partial Pressure; Retina; Sodium-Bicarbonate Symporters; Stilbenes; Tissue Distribution

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies have identified two oligomeric forms of band 3 whose proportions on gel profiles were modulated by the particular ligand occupying the intramonomeric stilbenedisulfonate site during intermonomeric cross-linking by BS3 [bis-(sulfosuccinimidyl) suberate] [Salhany et al. (1990) J. Biol. Chem. 265, 17688-17693]. When DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) was irreversibly attached to all monomers, BS3 covalent dimers predominated, while with DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) present to protect the intramonomeric stilbenedisulfonate site from attack by BS3, a partially cross-linked band 3 tetramer was observed. In the present study, we investigate the structure of the protected stilbenedisulfonate site within the tetrameric complex by measuring the ability of patent monomers to react irreversibly with DIDS. Our results show two main populations of band 3 monomers present after reaction with DNDS/BS3: (a) inactive monomers resulting from the displacement of reversibly bound DNDS molecules and subsequent irreversible attachment of BS3 to the intramonomeric stilbenedisulfonate site and (b) residual, active monomers. All of the residual activity was fully inhibitable by DIDS under conditions of reversible binding, confirming expectations that all of the monomers responsible for the residual activity have patent stilbenedisulfonate sites. However, within this active population, two subpopulations could be identified: (1) monomers which were irreversibly reactive toward DIDS and (2) monomers which were refractory toward irreversible binding of DIDS at pH 6.9, despite being capable of binding DIDS reversibly. Increasing the pH to 9.5 during treatment of DNDS/BS3-modified cells with 300 microM DIDS did not cause increased irreversible transport inhibition relative to that seen for cells treated at pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Macromolecular Substances; Protein Binding; Stilbenes

1. Ionic selectivity of an adrenaline-induced current was investigated in single guinea-pig ventricular cells by recording whole-cell currents using the patch clamp technique combined with internal perfusion. Other ionic currents and exchange currents known in ventricular cells were suppressed by appropriate inhibitors and the adrenaline-induced current was defined as a difference between currents obtained in the presence and absence of adrenaline. 2. The adrenaline-induced current was time independent and its I-V relation showed saturation of the inward current in the negative voltage range. 3. The reversal potential was approximately -20 mV with 140 mM-NaCl external solution and Cs(+)-rich internal solution containing 51 mM-Cl-. Replacing Na+ with various monovalent and divalent cations (Li+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+) produced no appreciable change in the reversal potential. 4. Varying the external Cl- concentration ([Cl-]o) in exchange for aspartate or benzenesulphonate greatly changed the reversal potential. The relationship between the reversal potential and log[Cl-]o indicated a slope of 59.5 or 53.6 mV per tenfold change in [Cl-]o in the presence of 51 or 102 mM-Cl- in the internal solution, respectively. 5. Anion substitutions did not appreciably affect the I-V relation before application of adrenaline, suggesting that the cell membrane had a low Cl- conductance in the control state. 6. 4.4'-Dinitrostilbene-2-2'-disulphonic acid (DNDS: 1-10 mM), a specific inhibitor of membrane chloride permeability, depressed the adrenaline-induced current without changing the reversal potential. 7. The results suggest strongly that the adrenaline-induced current is carried mainly by Cl-. However, the development of this current appears to depend also on external cations, since the magnitude of the adrenaline response varied depending on the external cations species, with no response in Tris-HCl or TEA-Cl solution. The external cations may facilitate the adrenaline response with a sequence of efficacy of Na+ greater than K+, Rb+ greater than Cs+, Li+, divalent cations.

Topics: Animals; Aspartic Acid; Benzenesulfonates; Cell Membrane Permeability; Chlorides; Epinephrine; Guinea Pigs; Ion Channels; Ion Exchange; Membrane Potentials; Myocardium; Stilbenes; Time Factors

Treatment of intact human erythrocytes with bis(sulfosuccinimidyl)suberate converted band 3 to two species with lower electrophoretic mobility in sodium dodecyl sulfate (SDS). The presence of the noncovalent anion transport inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonate, promoted the lowest mobility form, while a closely related analogue, 4,4'-diisothiocyano-2,2'-stilbenedisulfonate, did not. Ferguson analysis of the electrophoretic behavior of the two slowly migrating bands strongly suggested that they represented dimers and tetramers of band 3. Increasing the temperature of the SDS solution to greater than 60 degrees C quantitatively converted the tetrameric species to the dimeric form. We conclude that band 3 can be intermonomerically cross-linked by bis(sulfosuccinimidyl)suberate as covalent dimers within two alternate quaternary forms in a manner modulated by the ligand occupying the intramonomeric stilbenedisulfonate site. In one form, band 3 covalent dimers are noncovalently associated as a SDS-resistant tetramer, while in the other form, covalent dimers are not so associated. There is no obvious relationship between ligand stereochemistry and the resulting quaternary form, suggesting that the two forms reflect alternate allosterically modulated porter quaternary structures. The significance of these two quaternary states to the transport or the ankyrin binding functions of band 3 is unknown.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Cross-Linking Reagents; Humans; Kinetics; Ligands; Macromolecular Substances; Stilbenes; Succinimides; Thermodynamics

The phosphate self-exchange flux in resealed erythrocyte ghosts and in amphotericin B (5.5 microM) permeabilized erythrocytes has been studied. The phosphate self-exchange flux exhibits an S-shaped concentration dependence and a self-inhibition in permeabilized red cells while in erythrocyte ghosts no self-inhibition of the phosphate flux has been observed. The apparent half-saturation constants and the apparent Hill coefficients were assessed by the double reciprocal Hill plots of 1/JP versus 1/[P]n. The phosphate half-saturation constants amount to approx. 125 mM in ghosts and to about 75 mM in permeabilized cells while the apparent Hill coefficients amount to 1.15 and to 1.65 (pH 7.2, 25 degrees C), respectively. Both chloride and sulfate elicit a mixed-type inhibition of the phosphate self-exchange flux. In permeabilized cells, chloride and sulfate shift the flux optimum towards higher phosphate concentrations and reduce the apparent Hill coefficients. In erythrocyte ghosts, the apparent Hill coefficients are insensitive to these anions. The double reciprocal Hill plots indicate a mixed-type inhibition of the phosphate self-exchange flux by DNDS, salicylate and dipyridamole and a noncompetitive inhibition of the phosphate self-exchange flux by phlorhizin. By contrast, the Hill-Dixon plots for chloride and sulfate indicate a competitive inhibition of the phosphate self-exchange flux in erythrocyte ghosts and a mixed-type inhibition in permeabilized cells and provide Hill coefficients of greater than unity for chloride and sulfate. The Dixon plots for DNDS, salicylate, phlorhizin and dipyridamole show a noncompetitive inhibition of the phosphate flux and provide apparent Hill coefficients of 0.95-1.0 for inhibitor binding. Using the Debye-Hückel theory, the effects of ionic strength upon phosphate transport and inhibitor binding can be eliminated. The results of our studies provide strong evidence for the assumption that electrostatic forces are involved in phosphate transport and in inhibitor binding.

Topics: Adult; Biological Transport; Chlorides; Dipyridamole; Erythrocyte Membrane; Erythrocytes; Humans; Kinetics; Mathematics; Phlorhizin; Phosphates; Salicylates; Salicylic Acid; Stilbenes; Sulfates

The exit of HCO3- across the basolateral membrane of the proximal tubule cell occurs via the electrogenic cotransport of 3 eq of base per Na+. We have used basolateral membrane vesicles isolated from rabbit renal cortex to identify the ionic species transported via this pathway. Media of varying pH and pCO2 were employed to evaluate the independent effects of HCO3- and CO3(2-) on 22Na transport. Na+ uptake was stimulated when [CO3(2-)] was increased at constant [HCO3-], indicating the existence of a transport site for CO3(2-). In the presence of HCO3-, Na+ influx was stimulated more than 3-fold by an inward SO3(2-) gradient. SO3(2-)-stimulated Na+ influx was stilbene-sensitive, confirming that it occurs via the Na+-HCO3- cotransport system. Na+-SO3(2-) cotransport was demonstrated and found to have a 1:1 stoichiometry. Increasing [CO3(2-)] at constant [HCO3-] reduced the stimulation of Na+ influx by SO3(2-), suggesting competition between SO3(2-) and CO3(2-) at a common divalent anion site. Additional divalent anions that were tested, such as SO4(2-), oxalate2-, and HPO4(2-), did not interact at this site. SO3(2-) stimulation of Na+ influx was absolutely HCO3-(-)dependent and was increased as a function of [HCO3-], indicating the presence of a separate HCO3- site. Lastly, we tested whether Na+ interacts via ion pair formation with CO3(2-) or binds to a distinct site. Na+, which has lower affinity than Li+ for ion pair formation with CO3(2-), was found to have greater than 5-fold higher affinity than Li+ for the Na+-HCO3- cotransport system. Moreover, when its inhibition was studied as a function of [Na+], harmaline was found to be a competitive inhibitor of Na+ influx, indicating the existence of a distinct cation site. Our data are compatible with a model in which base transport across the basolateral membrane of the proximal tubule cell takes place via 1:1:1 cotransport of CO3(2-), HCO3-, and Na+ on distinct sites.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Bicarbonates; Binding, Competitive; Biological Transport; Carbon Dioxide; Carrier Proteins; Cell Membrane; Hydrogen-Ion Concentration; Kidney; Lithium; Male; Rabbits; Sodium; Sodium-Bicarbonate Symporters; Stilbenes; Sulfites

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.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Exchange Protein 1, Erythrocyte; Base Sequence; Chlorides; Dipyridamole; Erythrocytes; Female; Humans; Kinetics; Lysine; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Oocytes; Protein Binding; Stilbenes; Xenopus laevis

We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.

Topics: Animals; Chloride Channels; Chlorides; Colon; Electric Conductivity; Epithelium; Female; Kinetics; Lipid Bilayers; Membrane Proteins; Rats; Rats, Inbred Strains; Stilbenes

Nonselective Ca2+-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca2+ on the cytosolic side, the mean open-state probability Po of one channel was about 0.5. In inside-out oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenelpropylamino)-benzoic acid (NPPB) and 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mumol/l of NPPB or DPC decreased Po from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mumol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reduced Po, but in contrast to the above mentioned substances, it induced fast flickering. Ba2+ (70 mmol/l) and tetraethylammonium (TEA+; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitro-2,2'-stilbenedisulfonate (DNDS). 10 mumol/l SITS applied to the cytosolic side increased Po from 0.5 to 0.7 and with 100 mumol/l SITS the channels remained nearly permanently in its open state (Po approximately equal to 1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca2+ concentration to 0.1 mumol/l, addition of 100 mumol/l of SITS had no effect. Similarly, reducing bath Ca2+ concentration from 1.3 mmol/l in presence of 100 mumol/l SITS (channels are maximally activated) to 0.1 mumol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca2+. SITS had no effects, when applied to the extracellular side in out-side out patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Aniline Compounds; Animals; Calcium Channels; Cell Membrane; Cytosol; Diphenylamine; Nitrobenzoates; ortho-Aminobenzoates; Pancreas; Quinine; Rats; Stilbenes

To understand the route of HCO3- exit across the basolateral membrane of Amphiuma small intestinal cells during active H+ secretion the influence of bath HCO3- on serosal membrane potential (Vs) was measured using conventional microelectrodes. The villus sheet preparation was used, which allowed direct access to the basal membrane for microelectrode impalement. When tissues were incubated in Cl(-)-free (SO2-4 based) medium, Vs averaged -85.8 mV. Elevation of serosal bath [HCO3-] at constant CO2 increased Vs; reduction of the [HCO3-] reduced Vs and increased the fractional resistance of the serosal membrane. When serosal bath HCO3- and CO2 were completely replaced, Vs was -44.8 mV. The serosal membrane was also depolarized on complete replacement of medium HCO3- and CO2 when bath pH was buffered with phosphate. 4,4'-Dinitro-2,2'-stilbene disulfonate (DNDS) and acetazolamide blocked the decline in Vs produced by lowering serosal medium [HCO3-]. Replacement of serosal Na+ reduced Vs 32.7 mV. DNDS blocked this response. It is concluded that a Na+-dependent, stilbene-sensitive HCO3- exit pathway resides in the basolateral membrane of the intestinal cells. Possibly there is also a parallel, conductive pathway for HCO3-.

Topics: Acetazolamide; Animals; Bicarbonates; Carrier Proteins; Cell Membrane; Electric Conductivity; In Vitro Techniques; Intestinal Mucosa; Intestine, Small; Kinetics; Membrane Potentials; Muscle, Smooth; Sodium; Sodium-Bicarbonate Symporters; Stilbenes; Urodela

Diethyl pyrocarbonate inhibited the phosphate exchange across the human erythrocyte membrane. The exchange rate was inhibited only when the membranes were modified with the reagent from the cytosolic surface of resealed ghosts. The intracellular modification by diethyl pyrocarbonate inhibited the extracellular binding of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to band 3 protein. Furthermore, the extracellular 4,4'-dinitrostilbene-2,2'-disulfonic acid protected the membranes from the intracellular modification by diethyl pyrocarbonate. These results suggest that the extracellular binding of 4,4'-dinitrostilbene-2,2'-disulfonic acid to band 3 protein induces the conformational change of the intracellular counterpart of band 3 protein and the diethyl pyrocarbonate susceptible residue(s) is (are) hidden from the cytosolic surface of the cell membrane in connection with the conformational change. Conversely, under the conditions where the diethyl pyrocarbonate modification is confined to the intracellular side of the membrane, the extracellular binding site of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid is hidden from the cell surface.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amino Acids; Anion Exchange Protein 1, Erythrocyte; Biological Transport; Cytosol; Diethyl Pyrocarbonate; Erythrocyte Membrane; Formates; Humans; Hydrogen-Ion Concentration; Phosphates; Protein Conformation; Stilbenes; Surface Properties

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.

Topics: Animals; Axons; Bicarbonates; Decapodiformes; Hydrogen-Ion Concentration; In Vitro Techniques; Sodium; Stilbenes

The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Site; Anion Exchange Protein 1, Erythrocyte; Anions; Binding Sites; Binding, Competitive; Biological Transport; Chlorides; Dipyridamole; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Mathematics; Phosphates; Stilbenes; Sulfates

The transport inhibitor DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) changes the bis(sulfosuccinimidyl)suberate (BS3) crosslinking pattern of band 3 protein from a mixture of dimer-crosslinkable (DC) and tetramer-crosslinkable (TC) states to the TC-state as the exclusive crosslinked product for reactions occurring in membranes of intact human erythrocytes. Pretreatment of cells with DNDS followed by extensive washing restores the original DC to TC proportionality indicating that the two states are reversibly interconvertible. We suggest a model wherein band 3 transport site ligands allosterically modulate the global conformation of a tetrameric porter between two reversibly interconvertible quaternary structures. These transitions in quaternary structure may be important to transmembrane signaling of events between the exofacial ligand binding site and the sites on the porter extension which bind ankyrin and hemoglobin.

Topics: Allosteric Regulation; Allosteric Site; Anion Exchange Protein 1, Erythrocyte; Cross-Linking Reagents; Erythrocyte Membrane; Humans; Kinetics; Ligands; Macromolecular Substances; Models, Molecular; Protein Conformation; Stilbenes; Succinimides

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Carbodiimides; Citrates; Erythrocyte Membrane; Ethyldimethylaminopropyl Carbodiimide; Humans; In Vitro Techniques; Ion Channels; Stilbenes

In order to investigate the possibility that there may be two conformationally distinct dopamine D1 binding sites, the effect of lysine-modifying agents on striatal dopamine D1 receptors was investigated. Treatment with the distilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, (DIDS), resulted in an irreversible D1 receptor inactivation that was associated with a 70% loss of binding sites. The remaining DIDS-insensitive sites displayed both a decreased affinity (approximately 5 fold) for the D1 antagonist SCH-23390 and an enhanced affinity of dopaminergic agonists (approximately 10 fold) for the agonist high-affinity form of the receptor. Pretreatment with Gpp(NH)p, a non-hydrolysable guanine nucleotide, prevented the formation of the agonist high-affinity form, indicating that these sites are G-protein-linked. Prior occupancy of D1 receptors with dopaminergic agonists and antagonists afforded no protection against DIDS inactivation, suggesting that a site outside the ligand binding subunit of the D1 receptor was modified. Taken together, these data suggest that [3H]SCH-23390 labels two conformationally distinct populations of dopamine D1 receptors.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Brain; Brain Chemistry; Cell Membrane; Dogs; Dose-Response Relationship, Drug; Kinetics; Receptors, Dopamine; Receptors, Dopamine D1; Stilbenes

1. The inhibition of 36Cl efflux with stilbene disulphonates, SD, has been studied under conditions of chloride equilibrium in depolarized fibre bundles from frog semitendinosi. The chosen probes were the aminoreactive derivative SITS and the derivative DNDS with no aminoreactive group. SD were added to the medium during 36Cl efflux allowing the estimation of fractional inhibition after a single 36Cl loading. 2. Both probes inhibited chloride self-exchange reversibly within the pH range 5.5-9.5 under study. 3. At SD concentrations above the half-inhibition concentration the inhibition reached a steady level with a time lag equal to that required for extracellular fluid change. The time constant for reversibility upon the removal of SD increased with decreasing pH, but rapid reversibility always appeared upon an increase of pH to 7.2. These findings suggest that SD may enter the membrane at low pH, but that the inhibitory action is confined to superficial membrane sites. 4. The inhibitory power of both probes showed a pronounced pH dependence, pK approximately 7. The half-inhibition concentration increased about 6-7 times when pH was lowered one unit from the pK value. 5. The apparent affinity of SITS to the transport system was about 5 times higher than that of DNDS. The apparent dissociation constants at neutral pH were 8.5 x 10(-5) M (SITS) and 4.5 x 10(-4) M (DNDS). Both probes showed a maximal inhibition close to 100% at neutral pH and approximately 85% at pH 5.5. 6. The inhibition depended on the chloride concentration in a way consistent with competitive inhibition in both neutral and acid media. 7. The results are consistent with the classical model of anion transport in frog muscle, suggesting that SD and chloride may compete for binding to a site with increasing anion affinity upon protonation; the results do not, however, exclude that the conductive and the non-conductive chloride transport modes in frog muscle are mediated by separate SD-sensitive transport pathways.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Biological Transport; Chlorides; Depression, Chemical; Hydrogen-Ion Concentration; In Vitro Techniques; Muscles; Rana temporaria; Stilbenes; Time Factors

Passive K fluxes, measured with 86Rb, were investigated in osmotically swollen human erythrocytes. K influx and efflux increased progressively with increased hypotonicity up to 167 mosmol/kg. No increase in K flux was seen when NO3 or methylSO4 were substituted for Cl. Substitution of choline or N-methylglucamine for external Na reduced the K flux in swollen cells by only 22%, compared with a 60% reduction in euvolumic cells. However, the magnitude of this Na-dependent component was slightly, but significantly, higher in swollen cells. The presence of Na-dependent K influx in swollen cells was confirmed by measurements of Na influx demonstrating a K-dependent Na influx of similar magnitude in isovolumic and swollen cells. The volume-sensitive K flux was inhibited by bumetanide, but significantly less so than was Cl-dependent flux in isovolumic cells (half-maximal inhibition at 1.0 X 10(-4) vs. 5.8 X 10(-7) M). Kinetic analysis revealed that Cl-dependent K influx had a lower affinity for external K in swollen cells than in euvolumic cells (Km was 29.8 vs. 6.1 mM). The increased K flux in swollen cells was found to be transient, decreasing substantially and reverting back to a predominantly Na-dependent and more bumetanide-sensitive form after 2 h. The results indicate that swelling of human erythrocytes activates a transient Cl-dependent K flux that differs significantly from that in isovolumic cells in that it is less Na dependent, less sensitive to bumetanide, and has a lower affinity for K. Na-K cotransport is either unaffected or slightly increased in swollen cells. The altered flux in swollen cells would thermodynamically favor a volume-regulatory KCl efflux.

Topics: Biological Transport; Bumetanide; Chlorides; Erythrocytes; Humans; In Vitro Techniques; Ion Channels; Potassium; Sodium; Stilbenes; Water-Electrolyte Balance

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Chymotrypsin; Glutamates; Glutamic Acid; Humans; Isoxazoles; Membrane Proteins; Methylation; Molecular Weight; Stilbenes

Sickle cells exhibit a striking increase in monovalent cation content upon deoxygenation. These deoxy cation fluxes are reversible upon reoxygenation, balanced (Na flux = K flux), activated under physiologic conditions of pH and PO2, and insensitive to ouabain and furosemide. Our recent evidence indicates that deoxy cation fluxes and the Na/K pump, acting in concert, contribute to cation depletion in sickle cells in vitro. The resultant dehydration worsens their rigidity and increases their tendency to sickle upon deoxygenation. Here we report our finding that deoxy cation fluxes are inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). This irreversible inhibitor of anion transport was effective at concentrations less than 1 microM. The reversible, competitive inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) also blocked deoxy cation fluxes, at 100 microM. Niflumic acid, which exhibits non-competitive inhibitory kinetics against anion transport, also inhibited deoxy cation fluxes at 50 microM. Phloretin (a non-competitive anion transport inhibitor), however, had no effect on deoxy fluxes. On the basis of these data, we propose the working hypothesis that deoxygenation of sickle cells causes a conformational change in the anion exchange protein which allows for the passage of cations and alters its sensitivity to inhibitors.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anemia, Sickle Cell; Anion Transport Proteins; Carrier Proteins; Cell Membrane Permeability; Erythrocyte Membrane; Hemoglobin, Sickle; Humans; In Vitro Techniques; Niflumic Acid; Phloretin; Potassium; Sodium; Stilbenes

Chloride tracer efflux was measured from intact human erythrocytes into media containing different chloride concentrations and different concentrations of the inhibitors 4,4'-dinitrostilbene-2-2'-disulfonate (DNDS), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), phloretin, and sulfate. The data were analyzed to test whether these inhibitors were mutually exclusive with each other or whether they could bind at the same time. Under the assumption that mutual exclusiveness is due to steric interference, the data can be used to map out the protein surface near the outward-facing anion binding-transport site. It is concluded that there are separate domains for NAP taurine and phloretin that do not overlap with each other or with the chloride binding site. These two domains do, however, overlap with the binding domain for DNDS that, in addition, excludes the binding of chloride and sulfate.

Topics: Anions; Binding Sites; Binding, Competitive; Biological Transport; Chlorides; Erythrocytes; Humans; Phloretin; Stilbenes; Sulfates; Taurine

Phosphate entry into chloride-loaded human erythrocytes is inhibited by treatment of cells with the water-soluble carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the absence of added nucleophile. EAC does not penetrate the erythrocyte membrane or lead to significant intermolecular cross-linking of membrane proteins. At neutral extracellular pH in chloride-free medium, only about 50% of transport is rapidly and irreversibly inhibited, but at alkaline pH, inhibition is more rapid and complete. Inhibition by EAC was reversible in the presence of extracellular NaCl. Modification of membrane sulfhydryl groups does not prevent inhibition of phosphate transport by EAC but almost complete protection is afforded by 4,4-dinitrostilbene-2,2-disulfonic acid, a reversible competitive inhibitor of anion transport. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate, a reversible noncompetitive inhibitor of anion transport did not protect against EAC inhibition of transport but prevented reversal of inhibition in saline medium. Transport inhibition by [3H]EAC did not lead to specific incorporation of radioactivity into Band 3, the anion transport protein. These results suggest that inhibition of anion transport by EAC is due to modification of a carboxylic acid residue in or near the transport site accessible from the external face of the membrane. The subsequent fate of the modified carboxyl residue appears to be sensitive to the orientation of the anion transport site.

Topics: 4-Chloromercuribenzenesulfonate; Anions; Biological Transport; Carbodiimides; Cell Membrane Permeability; Chlorides; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; Humans; Hydrogen-Ion Concentration; Phosphates; Stilbenes; Taurine

Subunit interactions in the band 3 protein of the human red blood cell membrane have been examined by a combination of cross-linking, chemical labeling, and in situ proteolysis. In agreement with Staros (Staros, J. V. (1982) Biochemistry 21, 3950-3955), we find that the membrane-impermeant active ester bis(sulfosuccinimidyl) suberate (BSSS) cross-links band 3 in intact cells to a dimer, with no formation of higher oligomer. Combined cross-linking of the outer surface with BSSS and the cytoplasmic domain with Cu2+/o-phenanthroline does not produce significant covalent tetramer of band 3 (beyond that produced by Cu2+/o-phenanthroline alone). Therefore, the membrane domains and cytoplasmic domains of the same pair of subunits are cross-linked to each other. 4,4'-Diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) is known to form a covalent cross-link between complementary chymotryptic fragments (Mr 60,000 and 35,000). Edman degradation of band 3 from H2DIDS/chymotrypsin-treated cells shows that the H2DIDS cross-link is between fragments of the same subunit. In contrast, BSSS forms both intramolecular and intermolecular cross-links between complementary chymotryptic fragments. No intermolecular cross-links between two 35,000-dalton or two 60,000-dalton fragments are detectable. We have localized one end of the BSSS intermolecular cross-link to within 4 residues of the exofacial chymotrypsin cleavage site. The polypeptide sequence on each side of the site suggests that hydrophobic membrane-crossing segments emerge at the cell surface near the site of intermolecular cross-linking. This is the first detailed information available on the regions of the band 3 primary structure near the interface between subunits.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Chymotrypsin; Humans; Macromolecular Substances; Molecular Weight; Peptide Fragments; Protein Conformation; Stilbenes; Succinimides

35Cl NMR, which enables observation of chloride binding to the anion transport site on band 3, is used in the present study to determine the minimal structure containing the intact transport site. Removal of cytoskeletal and other nonintegral membrane proteins, or removal of the 40-kDa cytoskeletal domain of band 3, each leave the transport site intact. Similarly, cleavage of the 52-kDa transport domain into 17- and 35-kDa fragments by chymotrypsin leaves the transport site intact. Extensive proteolysis by papain reduces the integral red cell membrane proteins to their transmembrane segments. Papain treatment removes approximately 60% of the extramembrane portion of the transport domain and produces small fragments primarily in the range 3-7 kDa, with 5 kDa being most predominant. Papain treatment damages, but does not destroy, chloride binding to the transport site; thus, the minimal structure containing the transport site is composed solely of transmembrane segments. In short, the results are completely consistent with a picture in which the transport site is buried in the membrane where it is protected from proteolysis; the transmembrane segments that surround the transport site are held together by strong attractive forces within the bilayer; and the transport site is accessed by solution chloride via an anion channel leading from the transport site to the solution.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Bicarbonates; Binding Sites; Biological Transport; Chlorides; Chymotrypsin; Cytoskeleton; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Membrane Proteins; Osmolar Concentration; Papain; Peptide Fragments; Phenylglyoxal; Stilbenes

Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37 degrees C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37 degrees C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.

Topics: Binding Sites; Carbodiimides; CME-Carbodiimide; Erythrocytes; Ethyldimethylaminopropyl Carbodiimide; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Proteins; Phosphates; Stilbenes; Time Factors

Human erythrocytes were treated with various hydrophobic arylisothiocyanates under conditions which favor modification of distinct proteinaceous nucleophiles. The morphological appearance of phenylisothiocyanate-treated cells was discoid and membrane-bound hydrolases (human acetylcholinesterase, sheep phospholipase A2) were fully active following membrane modification. Noncharged hydrophobic arylisothiocyanates, including phenylisothiocyanate, beta-naphthylisothiocyanate and heterobifunctional azidoarylisothiocyanates inhibited [35S]-sulfate efflux irreversibly. Protection against modification-induced inhibition of sulfate transport was attained by the simultaneous presence of the specific reversible anion transport inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate. Selective protection of a functionally relevant domain of band 3 is concluded to occur based on the above-derived information.

Topics: 1-Naphthylisothiocyanate; Acetylcholinesterase; Anion Exchange Protein 1, Erythrocyte; Anion Transport Proteins; Carrier Proteins; Erythrocyte Membrane; Humans; Isothiocyanates; Phospholipases A; Phospholipases A2; Stilbenes; Sulfates; Thiocyanates

Band 3 is an integral membrane protein that exchanges anions across the red cell membrane. Due to the abundance and the high turnover rate of the band 3 transport unit, the band 3 system is the most heavily used ion-transport system in a typical vertebrate organism. Here we show that 35Cl NMR enables direct and specific observation of substrate Cl- binding to band 3 transport sites, which are identified by a variety of criteria: (a) the sites are inhibited by 4,4'- dinitrostilbene -2,2'-disulfonate, which is known to inhibit competitively Cl- binding to band 3 transport sites; (b) the sites have affinities for 4,4'- dinitrostilbene -2,2'-disulfonate and Cl- that are quantitatively similar to the known affinities of band 3 transport sites for these anions; and (c) the sites have relative affinities for Cl-, HCO-3, F-, and I- that are quantitatively similar to the known relative affinities of band 3 transport sites for these anions. The 35Cl NMR assay also reveals a class of low affinity Cl- binding sites (KD much greater than 0.5 M) that are not affected by 4,4'- dinitrostilbene -2,2'-disulfonate. These low affinity sites may be responsible for the inhibition of band 3 catalyzed anion exchange that has been previously observed at high [Cl-]. In the following paper the 35Cl NMR assay is used to resolve the band 3 transport sites on opposite sides of the membrane, thereby enabling direct observation of the transmembrane recruitment of transport sites.

Topics: Anion Exchange Protein 1, Erythrocyte; Anions; Binding Sites; Biological Transport, Active; Chlorides; Erythrocyte Membrane; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mathematics; Models, Biological; Protein Binding; Stilbenes

Numerous models describing anion exchange across the red cell membrane by band 3 have been discussed in literature. These models are readily distinguished from one another by an experiment which tests the ability of band 3 transport sites to be recruited to one side of the membrane. In order to observe directly the transmembrane recruitment of transport sites, we have developed 35Cl NMR techniques that resolve the two transport site populations on opposite sides of the membrane. Using these techniques, we show that the inhibitors 4,4'- dinitrostilbene -2,2'-disulfonate and p- nitrobenzensulfonate each recruit all of the transport sites on both sides of the membrane to the extracellular facing conformation. This result indicates that band 3 has an alternating site transport mechanism: each band 3 transport unit possesses a single functional transport site which is alternately exposed first to one side of the membrane then to the other.

Topics: Anion Exchange Protein 1, Erythrocyte; Binding, Competitive; Biological Transport, Active; Chlorides; Erythrocyte Membrane; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mathematics; Models, Biological; Stilbenes

Transport of pyridoxal 5-phosphate (PLP) into erythrocytes was inhibited by inhibitors of anion transport including stilbene disulfonate compounds, indicating that it is mediated by Band 3 protein. When erythrocytes were treated with PLP and large amounts of free lysine and NaBH4, two membrane-spanning fragments of Band 3 (Mr = 17,000 and 35,000) were specifically labeled. When the cells were pretreated with 4,4'-dinitrostilbene 2,2'-disulfonate, the labeling in the 35,000-dalton fragment was inhibited. Erythrocytes labeled by PLP in both the 17,000- and 35,000-dalton fragments transported PLP at a decreased rate, whereas the cells labeled in only the 17,000-dalton fragment had essentially the same transport activity as the control when 4,4'-dinitrostilbene 2,2'-disulfonate was removed. The extent of inhibition of transport of inorganic phosphate in the labeled cells was similar to that of PLP. The results indicate that the 35,000-dalton fragment participates in the anion transport of the cell membrane.

Topics: Affinity Labels; Anion Exchange Protein 1, Erythrocyte; Biological Transport, Active; Blood Proteins; Erythrocytes; Humans; Molecular Weight; Pyridoxal Phosphate; Stilbenes

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal: arg stoichiometry of 2:1. Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were above 10. Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments. The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 10 micrometers, only 50% of the band 3 molecules were labeled with [3H]-DIDS during 90 min at 38 degrees C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Aldehydes; Anions; Arginine; Carrier Proteins; Chlorides; Erythrocyte Membrane; Erythrocytes; Extracellular Space; Humans; Hydrogen-Ion Concentration; Intracellular Fluid; Phenylglyoxal; Potassium Chloride; Stilbenes

The kinetics of human red blood cell Cl transport have been studied under nonequilibrium conditions to determine whether or not an outward Cl gradient can recruit the transport protein from an inward-facing to an outward-facing configuration. Three kinds of evidence are consistent with this outward recruitment. First, the initial net Cl efflux into a Cl-free phosphate medium is independent of the intracellular Cl concentration in the range 20-170 mM. Second, an outward Cl gradient strongly enhances the inhibitory potency of DNDS (4,4'-dinitro-2,2'-stilbene disulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, we have estimated the number of Cl ions transported during the putative outward recruitment. Resealed red cell ghosts containing only 70 muM 36Cl were resuspended at 0 degrees C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, or approximately 10(6) Cl ions per ghost, followed by a much slower further loss of Cl. The rapid loss of 10(6) Cl ions per ghost, which is abolished by pretreatment with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), appears to represent the Cl that is transported during the first half-turnover of the transport cycle. These data are strong evidence that the influx and efflux events in the catalytic cycle for anion transport do not take place simultaneously, and that the stoichiometry of the transport cycle is close to one pair of anions exchanged per band 3 monomer.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Biological Transport; Blood Proteins; Catalysis; Chemical Phenomena; Chemistry; Chlorides; Erythrocytes; Humans; Ion Channels; Ion Exchange; Mathematics; Phosphates; Stilbenes

Chloride exchange in resealed human erythrocyte ghosts can be irreversibly inhibited with phenylglyoxal, a reagent specific for the modification of arginyl residues in proteins. Phenylglyoxal inhibits anion transport in two distinct ways. At 0 degrees C, inhibition is instantaneous and fully reversible, whereas at higher temperature in an alkaline extracellular medium, covalent binding of phenylglyoxal leads to an irreversible inhibition of the transport membranes system. Indiscriminate modification of membrane arginyl residues was prevented by reacting the with phenylglyoxal in an alkaline extracellular medium while maintaining intracellular pH near neutrality. The rate of modification of anion transport depends on phenylglyoxal concentration, pH, temperature, and the presence of anions and reversible inhibitors of the anion transport system in fashions that are fully compatible with the conclusion that phenylglyoxal modifies arginyl residues that are essential for anion binding and translocation. Phenylglyoxal reacts rapidly with the deprotonated form of the reactive groups. It is proposed that the effects of anions and of negatively charged transport inhibitors on the rate of irreversible binding of phenylglyoxal are related to the effects of the anions on a positive interfacial potential. This potential determines the local pH, and thereby the concentration of deprotonated groups, in an exofacial region of the anion transport protein.

Topics: Aldehydes; Anions; Biological Transport; Chlorides; Cresols; Erythrocyte Membrane; Erythrocytes; Extracellular Space; Glucose; Humans; Hydrogen-Ion Concentration; Ion Channels; Phenylglyoxal; Salicylates; Stilbenes; Temperature

1. Dansylation of the red cell membrane produces inverse effects on SO(4) (2-) and Cl(-) equilibrium exchange. The former is enhanced by several orders of magnitude (Legrum, Fasold & Passow, 1980), the latter is inhibited. Both effects are potentiated after dansylation in the presence of 2-(4-amino-3-sulphophenyl)-6-methyl-7-benzothiazol sulphonic acid (APMB), a disulphonic acid that combines non-covalently with the 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulphonic acid (H(2)DIDS) binding site of the anion transport protein.2. After dansylation the maximum of the pH dependence of SO(4) (2-) exchange near pH 6.3 is replaced by a plateau. When dansylation is performed in the presence of APMB, the plateau is reached at a much higher level at around pH 7.0 and resembles that observed by Funder & Wieth (1976) for Cl(-).3. The mutual interactions between the transfer site, the H(2)DIDS binding site, and the as yet unidentified danysl chloride binding sites were studied in detail. Occupation of the H(2)DIDS binding site by the non-covalently binding agents 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS), 4,4'-bis(acetamido) stilbene-2,2'-disulphonate (DAS) or APMB inhibit the enhanced SO(4) (2-) exchange across the previously dansylated membrane. The apparent K(I) value remains the same as in untreated membranes for DNDS, is reduced to 1/3 for DAS, and to 1/60 for APMB. Conversely, when dansylation is carried out while the H(2)DIDS binding site is occupied by DNDS, APMB or DAS, the enhancement of SO(4) (2-) exchange (as measured after removal of excess dansyl chloride and the additional agent) is prevented by DNDS, augmented by APMB and not affected by DAS. This suggests that the agents stabilize different conformations of the H(2)DIDS binding site that are associated with different accessibilities of the dansyl chloride binding sites.4. The SO(4) (2-) equilibrium exchange as measured at a fixed Cl(-) concentration is enhanced when the Cl(-) concentration at which dansylation is carried out is increased, indicating allosteric interactions between anion binding and the exposure of the dansyl chloride binding sites.5. The enhanced K(+) efflux from dansylated red cells is independent of the described modifications of the dansylation reaction by APMB, DAS or DNDS, demonstrating that there exists no simple correlation between the changes of anion and cation movements that are induced by dansylation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Benzothiazoles; Binding Sites; Biological Transport; Chlorides; Dansyl Compounds; Erythrocyte Membrane; Erythrocytes; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Stilbenes; Sulfates; Thiazoles

The interaction between chloride and the anion transport inhibitor DNDS (4,4'-dinitro stilbene-2,2'-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cli and Clo. DNDS inhibited competitively the Clo-stimulated chloride efflux from intact red cells at 0 degree C and pH 7.8 with an inhibitor constant of 90 nM. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5 X 10(5) molecules/cell. Clo competitively inhibited DNDS binding with an inhibitor constant of 6 mM. In the absence of Clo the DNDS binding constant was 84 mM. The competition between chloride and DNDS was also tested in nystatintreated cells in which Clo always equaled Cli. Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20 degree C. We found that neither the DNDS binding constant nor the Clo inhibitor constant were significantly changed compared to 0 degree C.

Topics: Anion Transport Proteins; Biological Transport, Active; Carrier Proteins; Chlorides; Erythrocyte Membrane; Erythrocytes; Humans; Kinetics; Mathematics; Membrane Proteins; Models, Biological; Stilbenes