4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

To explore the impact of chloride ion channel and its blockers 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), cyanato-stilbene-2, 2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB) on arrhythmias caused by myocardial ischemia reperfusion.. A total of 40 rabbits were divided into control, ischemia reperfusion, DIDS low-dose, DIDS high-dose, SITS low-dose, SITS high-dose, NPPB low-dose and NPPB high-dose groups. Myocardial ischemia reperfusion model was established by ligation of anterior descending coronary artery. And standard limb lead II of electrocardiogram (ECG) was continuously monitored during the experimental process. Then comparisons of heart rate, ECG P wave, R wave, T wave, ST segment changes and arrhythmias score were made between the above groups.. During 30-minute ischemia, compared with the control group, all other groups showed significantly decreased heart rate ((199.8 ± 4.0) - (253.6 ± 2.1) vs (267.0 ± 3.4), all P < 0.01), elevated ECG P wave ((0.216 ± 0.019) - (0.356 ± 0.024) vs (0.186 ± 0.019), all P < 0.01), R wave ((0.564 ± 0.017) - (1.138 ± 0.048) vs (0.506 ± 0.018), all P < 0.01), T wave ((0.542 ± 0.013) - (0.856 ± 0.045) vs (0.278 ± 0.015), all P < 0.01) and ST segment ((0.326 ± 0.027) - (0.668 ± 0.054) vs (0.024 ± 0.023), all P < 0.01) and increased arrhythmia score ((1.4 ± 0.5) - (4.6 ± 0.5) vs (0.4 ± 0.5), all P < 0.01). Compared with the ischemia reperfusion group, the above indices significantly improved in the intervention groups (heart rate: (214.8 ± 3.4) - (246.8 ± 4.0) vs (199.8 ± 4.0), all P < 0.01; P wave: (0.216 ± 0.019) - (0.316 ± 0.011) vs (0.356 ± 0.024), all P < 0.01; R wave: (0.564 ± 0.017) - (0.980 ± 0.035) vs (1.138 ± 0.048), all P < 0.01; T wave: (0.542 ± 0.013) - (0.792 ± 0.026) vs (0.856 ± 0.045), all P < 0.01; ST segment: (0.326 ± 0.027) - (0.596 ± 0.018) vs (0.668 ± 0.054), all P < 0.01; arrhythmia score: (1.4 ± 0.5) - (3.8 ± 0.4) vs (4.6 ± 0.5), all P < 0.01). Among which, the DIDS group was the best, followed by the SITS group and then the NPPB group. And the high-dose subgroups were better than those of the low-dose subgroups. During 60-minute reperfusion, the decreased heart rate upswing significantly in each group and the elevated P wave, R wave, T wave and ST segment fell back gradually. The DIDS group showed the most obvious amplitude change, followed by the SITS group and then the NPPB group. And the high-dose subgroups were better than those of the low-dose subgroups. The arrhythmia score during reperfusion showed the same trend.. Chloride ion channel is involved in the generation of myocardial ischemia reperfusion arrhythmia.Early application of chloride ion channel blockers DIDS, SITS and NPPB may improve the ECG manifestations and reduce the degree of reperfusion arrhythmia.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Chloride Channels; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitrobenzoates; Rabbits

Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro; however, it did not modify membrane potential in mature neurons. Furthermore, in immature cells, AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50mM K(+). Preincubation of the neurons with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride, or ouabain before addition of AZA-1 decreased the AZA-1-induced neurotoxicity and the increase in phosphorylated c-Jun-N-terminal kinase (JNK) caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anions; Cell Size; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Ion Channels; JNK Mitogen-Activated Protein Kinases; Marine Toxins; Membrane Potentials; Membrane Transport Modulators; Mice; Neurons; Nitrobenzoates; Ouabain; Phosphorylation; Potassium; Signal Transduction; Sodium-Hydrogen Exchangers; Spiro Compounds; Time Factors

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume-activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of approximately 75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(- )> Cl(- )> F(-)). Staurosporine (200 nM) did not block the activation of I(Cl,swell). The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 microM), SITS (200 microM), ATP (500 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 100 miccroM), and Suramin (10 microM) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(do) and fractional distance of the binding site (delta) of 334 microM and 47 %, 880 microM and 35 %, 2,100 microM and 49%, 188 microM and 27%, and 66.5 microM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I(Cl,swell) in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adenosine Triphosphate; Animals; Cell Membrane Permeability; Cell Size; Chloride Channels; Chlorides; Cyclic AMP; In Vitro Techniques; Ion Channel Gating; Kinetics; Leydig Cells; Male; Membrane Potentials; Mice; Nitrobenzoates; Osmotic Pressure; Protein Kinase Inhibitors; Protein Kinases; Pyridoxal Phosphate; Staurosporine; Suramin

Cottonseed (Gossypium sp.) meals are protein rich and inexpensive, but the presence of the polyphenolic dialdehyde, gossypol, is responsible of many toxic effects in animals including fishes. Recently an effect on the transepithelial ion transport in rat colon has been demonstrated. In this study we investigated the effect of gossypol on the transepithelial electrical parameters of the isolated intestine of seawater adapted eel, Anguilla anguilla, by employing a Ussing chamber technique. We showed that the addition of gossypol to the perfusion media reduced short circuit current (I(sc)), a measure of Cl- active absorption in this tissue, and increased tissue conductance (g(t)). The observation that the effect of gossypol on both I(sc) and g(t) was modified by the pretreatment with TFP, a calmodulin inhibitor, suggests that the substance acts via a Ca2+ calmodulin pathway and excludes the possibility that the observed effects were due to a cytotoxic action. In addition, experiments performed in the presence of verapamil suggest that the polyphenolic pigment increases Ca2+ influx. It is likely that gossypol stimulates a basolateral quinine sensitive K+ conductance producing a K+ flux in absorptive direction that explains the reduction of I(sc). In addition dilution potential experiments showed that the polyphenolic aldehyde increases the anion conductance of the paracellular pathway. In conclusion our study suggests that gossypol alters ion transport in eel intestine by acting on both transcellular and paracellular pathways. Since the intestine is an important organ for maintaining the water and ion balance in seawater adapted fish, it is conceivable that gossypol could impair the ability of the animals to adapt to the environment.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adaptation, Physiological; Anguilla; Animals; Buffers; Calcium; Electric Conductivity; Epithelial Cells; Glyburide; Gossypol; In Vitro Techniques; Intestines; Ion Channel Gating; Ion Transport; Mucous Membrane; Nitrobenzoates; Perfusion; Quinine; Seawater; Serous Membrane

Apoptosis of rat cardiomyocytes induced by staurosporine is prevented by a stilbene derivative (DIDS), which is a known blocker of both Cl(-)/HCO(3)(-) exchangers and Cl(-) channels. To clarify its target, staurosporine-induced activation of caspase-3, DNA laddering and cell death were examined in cultured rat cardiomyocytes. Removal of ambient HCO(3)(-), which minimizes the function of Cl(-)/HCO(3)(-) exchangers, failed to affect the preventive effect of DIDS on apoptosis. A carboxylate analog Cl(-) channel blocker, which does not block Cl(-)/HCO(3)(-) exchangers, also inhibited apoptotic events. Thus, rescue by DIDS of cardiomyocytes from apoptosis is mediated by blockage of Cl(-) channels.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Apoptosis; Bicarbonates; Caspase 3; Caspases; Chloride Channels; DNA Fragmentation; Myocytes, Cardiac; Nitrobenzoates; Potassium Channel Blockers; Rats; Staurosporine

We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)E(2). PGE(2) (>10 nM) stimulated an outwardly rectifying Cl(-) current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PGE(2)-induced Cl(-) current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was I(-) > Br(-) approximately Cl(-) > gluconate(-). When outwardly rectifying Cl(-) current was induced by hyposmotic extracellular solution, no further stimulatory effect of PGE(2) was seen. Forskolin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PGE(2). The PGE(2)-induced Cl(-) current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDPbetaS), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PGE(2) (1 microM) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PGE(2) is known to exert its effects through four subtypes of PGE receptors (EP1-EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PGE(2) on osteoclasts. Our results show that PGE(2) stimulates rat osteoclast Cl(-) current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Cell Movement; Chloride Channels; Cyclic AMP; Dinoprostone; Electric Conductivity; Intracellular Membranes; Membrane Potentials; Nitrobenzoates; Osmosis; Osteoclasts; Permeability; Rats; Rats, Wistar; Receptors, Prostaglandin; Signal Transduction

K+ and Cl- homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K+ and Cl- efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl- current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C2-ceramide, or serum deprivation. Cl- channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl- channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K+ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl- and K+ homeostasis in regulating different apoptotic events.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Apoptosis; Caspases; Cell Size; Cells, Cultured; Cerebral Cortex; Chloride Channels; Enzyme Inhibitors; Membrane Potentials; Mice; Microscopy, Electron, Transmission; Neurons; Nitrobenzoates; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels

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

The Cl- channel blocker NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid) inhibited photosynthetic oxygen evolution of isolated thylakoid membranes in a pH-dependent manner with a K(i) of about 2 microM at pH 6. Applying different electron acceptors, taking electrons either directly from photosystem II (PS II) or photosystem I (PS I), the site of inhibition was localized within PS II. Measurements of fluorescence induction kinetics and thermoluminescence suggest that the binding of NPPB to the QB binding site of PS II is similar to the herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). The effects of different arylaminobenzoate derivatives and other Cl- channel inhibitors on photosynthetic electron transport were investigated. The structure--activity relationship of the inhibitory effect on PS II shows interesting parallels to the one observed for the arylaminobenzoate block of mammalian Cl- channels. A molecular modeling approach was used to fit NPPB into the QB binding site and to identify possible molecular interactions between NPPB and the amino acid residues of the binding site in PS II. Taken together, these data give a detailed molecular picture of the mechanism of NPPB binding.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Binding, Competitive; Chloride Channels; Electron Transport; Herbicides; Hydrogen-Ion Concentration; Kinetics; Luminescent Measurements; Models, Molecular; Nitrobenzoates; Oxygen; Photolysis; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Photosystem II Protein Complex; Pisum sativum; Spinacia oleracea; Structure-Activity Relationship; Thylakoids

Eel intestinal epithelium when bathed symmetrically with normal Ringer solution develops a net Cl(-) current (short circuit current, Isc) giving rise to a negative transepithelial potential (Vt) at the basolateral side of the epithelium, lower in fresh-water (FW)-acclimated animals with respect to sea-water (SW). The aim of the present work was to study the cell response to hypertonic stress of FW eel intestinal epithelium in relation to Cl(-) absorption. The hypertonicity of the external bathing solutions produced first a transient increase of Vt and Isc, then followed (after 10-15 min) by a gradual and sustained increase which reached the maximum value after 40-60 min. The morphometric analysis of the intestine revealed the shrinkage of the cells after 5 min hypertonicity exposure, and then a regulatory volume increase (RVI) response, which parallels the gradual and sustained increase in the electrophysiological parameters. This last phase is inhibited by drugs known to block Cl(-) absorption in eel intestine, such as luminal bumetanide (10 microM), specific inhibitor of Na(+)-K(+)-2Cl(-) cotransport, or basolateral NPPB (0.5 mM), dichloro-DPC (0.5 mM), inhibitors of basolateral Cl(-) conductance. Serosal dimethyl-amiloride (100 microM), specific inhibitor of the Na(+)/H(+) antiport, was ineffective on the hyperosmotic response. Bicarbonate revealed a crucial role as a modulator of hypertonicity response, since in bicarbonate-free conditions or in the presence of serosal 0.25 mM SITS, blocker of HCO(3)(-) transport systems, the Isc response to hypertonicity was lost. In nominally Ca(2+)-free conditions the Isc response to hypertonicity was abolished. The same results were obtained by bilateral addition of 100 microM verapamil or 50 microM nifedipine or 1 mM lanthanum, known Ca(2+) channel blockers, indicating that extracellular Ca(2+) plays a key role for the activation of Cl(-) current in the response to hypertonic stress. The data show that in the eel intestinal epithelium the hypertonicity of the external medium affects cell volume which in turn might represent the signal to increase the rate of Cl(-) transport. This response is sustained by the activation of the luminal Na(+)-K(+)-2Cl(-) cotransporter and the functionality of basolateral Cl(-) channels.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adaptation, Physiological; Amiloride; Anguilla; Animals; Bumetanide; Calcium Channel Blockers; Carrier Proteins; Cell Polarity; Cell Size; Chlorides; Diphenylamine; Hydrogen-Ion Concentration; In Vitro Techniques; Intestinal Mucosa; Mannitol; Membrane Potentials; Nitrobenzoates; Osmolar Concentration; Potassium; Sodium; Sodium-Potassium-Chloride Symporters; Water; Water-Electrolyte Balance

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antibodies, Anti-Idiotypic; Calcium; Chloride Channels; Histamine Release; Immunoglobulin E; Mast Cells; Nitrobenzoates; Ovalbumin; p-Methoxy-N-methylphenethylamine; Peritoneum; Rats; Rats, Sprague-Dawley

To investigate the effects of inhibitors of chloride channels on lens volume and tissue architecture under isotonic conditions.. Rat lenses were maintained in organ culture under isotonic conditions in the presence of various putative chloride channel inhibitors. The effect of an inhibitor on lens wet mass and tissue morphology was determined by weighing and histologic examination, respectively.. Exposure to 100 microM of either 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB) or 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) caused an increase in wet mass and severe tissue disruption in the lens equatorial region. Two distinctly different zones of tissue damage were evident: a peripheral zone of fiber cell swelling and an inner zone of extensive tissue breakdown. Extracellular space dilations caused the extensive tissue damage in the inner zone and preceded the peripheral fiber cell swellings. That the observed effects were a consequence of the inhibition of chloride channels was supported by (1) the effectiveness of NPPB at the lower dose of 10 microM, (2) the absence of any NPPB effect in chloride-free medium, and (3) an identical effect after exposure to tamoxifen, an inhibitor of the chloride channel regulator p-glycoprotein.. Study results indicate that chloride channels are active in the lens under isotonic conditions. The spatial and temporal pattern of morphologic changes that was observed is consistent with a steady state efflux of chloride ions and water from peripheral fiber cells and a corresponding influx into fiber cells deeper in the lens. These observations may therefore represent the first visualization of the chloride flux postulated by others to be a component of the lens internal circulation system.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Body Water; Chloride Channels; Chlorides; Lens, Crystalline; Microscopy, Confocal; Nitrobenzoates; Organ Culture Techniques; Organ Size; Rats; Rats, Wistar; Tamoxifen

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

A stretch-activated Cl- current (ICl) was investigated in cultured murine microglia using the whole-cell configuration of the patch-clamp technique. After application of membrane stretch, a Cl- current appeared within seconds, and its amplitude increased further within 3-8 min. ICl underwent rundown, which was prevented by addition of 4 mM ATP to the intracellular perfusing solution. The stretch-activated Cl- current exhibited outward rectification and did not show any voltage-dependent gating. Lowering the concentration of extracellular Cl- from 142 to 12 mM by equimolar substitution of Cl- with gluconate shifted the reversal potential of ICl by 41.6 +/- 1.8 mV in the depolarizing direction. 4, 4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) blocked ICl in a voltage- and time-dependent manner. At a test potential of +40 mV, a half-maximal blockade at 16.1 microM DIDS and at 71.0 microM SITS was determined for ICl. At a concentration of 200 microM, 5-nitro-2-(3-phenylpropylamino)benzoic acid or flufenamic acid blocked ICl by 88% and 75%, respectively. Each of these four Cl- channel blockers reversibly inhibited the ramification process of microglia, whereas blockers of voltage-gated Na+ and K+ channels did not affect the transformation of microglia from their ameboid into the ramified phenotype. It is suggested that in microglia functional stretch-activated Cl- channels are required for the induction of ramification but not for maintaining the ramified shape.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; Brain Chemistry; Cells, Cultured; Chloride Channels; Culture Media, Conditioned; Flufenamic Acid; Macrophages; Membrane Potentials; Mice; Mice, Inbred Strains; Microglia; Nitrobenzoates; Patch-Clamp Techniques; Physical Stimulation; Stress, Mechanical; Tetrodotoxin

The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob, J. Physiol, Lond. 491: 743-755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTP gamma S-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C. and inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTP gamma S but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cattle; Chloride Channels; Chlorides; Ciliary Body; Electric Conductivity; Epithelium; Guanosine 5'-O-(3-Thiotriphosphate); Hypotonic Solutions; Nitrobenzoates; Phospholipases; Phospholipases A; Phospholipases A2; Pigmentation; Second Messenger Systems

Neuronal and glial cell swelling occurs rapidly in ischemia as part of the cytotoxic response. Astrocytic swelling is known to result in large extracellular increases in certain amino acids, including glutamate, aspartate and taurine, as part of the regulatory volume decrease (RVD) response inherent to these and other cells. RVD in astrocytic cultures is inhibited by anion channel blockers. In this study, we compared the effects of three anion channel blockers on the ischemia/reperfusion-evoked release of amino acids from the in vivo rat cerebral cortex. Twenty minutes of four vessel cerebral ischemia caused significant increases in cortical superfusate levels of aspartate, glutamate, GABA, taurine and phosphoethanolamine. During reperfusion there were delayed increases in the level of glycine, alanine and serine. Glutamine levels were not affected. Cl- channel blockers, 4-acetamido-4'-isothiocyanostrilbene-2,2'-disulfonic acid (SITS, 2 mM), 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB, 350 microM) and dipyridamole (200 microM) depressed basal releases of glutamate and taurine and the ischemia/reperfusion-evoked releases of aspartate, glutamate, taurine and phosphoethanolamine. The results suggest that diffusion of amino acids through an anion channel may be partially responsible for the elevated extracellular levels of excitotoxic and other amino acids that occur during cerebral ischemia/reperfusion.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amino Acids; Animals; Aspartic Acid; Brain Ischemia; Cerebral Cortex; Glutamic Acid; Ion Channels; Male; Nitrobenzoates; Rats; Rats, Sprague-Dawley

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

1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cattle; Chloride Channels; Ciliary Body; Cytoskeleton; Epithelial Cells; Epithelium; Fluorescent Antibody Technique, Direct; Hypotonic Solutions; In Vitro Techniques; Membrane Potentials; Nitrobenzoates; Patch-Clamp Techniques

Relationships between short-circuit current (Isc), cell Cl and the mechanism(s) of Cl accumulation in toad bladder epithelial cells were investigated. In serosal Cl-free gluconate Ringer, 80% of the cell Cl (measured by x-ray microanalysis) was lost over 30-60 min with an associated decrease in cell water content. concomitantly, Isc fell to 20% of its initial value within 10 min but then recovered to 45% of its initial value despite continued Cl loss. With the reintroduction of Cl, cell Cl and Isc both recovered within 10 min. Serosal SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate; 0.5 mM) plus bumetanide (0.1 mM), did not prevent the fall in Isc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of Isc in this medium. They also prevented the recovery of Isc in Cl medium but not the reaccumulation of Cl by the cells. Although SITS and bumetanide did not prevent the loss or recovery of Cl, they modified the pattern of the ion changes. In their absence, changes in cellular Cl were twice that of the changes in measured cellular cations implicating basolateral Cl/HCO3 exchange in Cl movement. With SITS plus bumetanide present, changes of similar magnitude in Cl were associated with equivalent changes in cation, consistent with the inhibition of Cl/HCO3 exchange.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Antiporters; Bendroflumethiazide; Bufo marinus; Bumetanide; Cell Size; Chloride-Bicarbonate Antiporters; Chlorides; Culture Media; Electron Probe Microanalysis; Epithelial Cells; Epithelium; Gluconates; Hydrochlorothiazide; Intracellular Fluid; Membrane Potentials; Nitrobenzoates; Sodium; Urinary Bladder

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K+ and Cl- containing solutions. An outward current detected at 0 mV, corresponded to a K+ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca(2+)-activated K+ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl- current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4'-isothiocyanostilbene), and its reversal potentials corresponded to the Cl- equilibrium potentials at high and low external Cl- concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K+ and Cl- currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K+ and Cl- conductances in these cells, and that the activation pathway for the K+ conductance apparently involves [Ca2+], while the activation pathway for the Cl- conductance does not involve [Ca2+] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Actins; Arachidonic Acid; Calcium; Cell Line; Cell Membrane; Chloride Channels; Cytochalasin D; Cytoskeleton; Dose-Response Relationship, Drug; Egtazic Acid; Guanosine Triphosphate; Humans; Hypotonic Solutions; Nitrobenzoates; Patch-Clamp Techniques; Potassium Channels; Quinacrine; Submandibular Gland

This report describes chloride and iodide effluxes across the basolateral membrane of porcine thyroid follicles reconstituted in culture. Basolateral chloride efflux is activated by thyrotropin (TSH). TSH (10 mU/ml) induces a twofold increase in the initial rate of chloride efflux. Forskolin (FSK, 5 microM) which increases intracellular cAMP also stimulates the initial rate of chloride efflux 3.5-fold, whereas an increase in the free cytosolic Ca2+ with the ionophore A23187 or thapsigargin, fails to mimic the TSH effect. The chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) dose dependently inhibits chloride efflux rates with the maximal and half maximal effects observed for 100 microM and 30 microM, respectively. Basolateral chloride efflux rates are also inhibited in the presence of the organic anion transporter blocker probenecid (5 mM) or the Cl-/HCO3- exchanger blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 250 microM), respectively, by 60% and 40%, whereas it is not affected by ClO4 (100 microM). The initial rate of iodide efflux is weakly activated (1.4-fold) by TSH (10 mU/ml). TSH effect could be reproduced by agents known to activate Ca(2+)-dependent processes as A23187, ionomycin (1 microM), phorbol 12-myristate 13-acetate (TPA, 0.1 microM) and epidermal growth factor (EGF, 0.1 microM) which increase the initial rate of iodide efflux from 1.2- to 1.8-fold, whereas FSK is without effect. The chloride channel blocker NPPB (500 microM) is required to significantly inhibit the initial rate of iodide efflux by 30%. The initial rate of iodide efflux is also reduced by 30% in the presence of SITS (250 microM) or probenecid (5 mM) whereas it is activated by ClO4 (100 microM). We conclude that basolateral chloride and iodide effluxes are both regulated by TSH, using two different transduction pathways. Chloride efflux regulation may involve a cAMP transduction signal, whereas the regulation of iodide efflux may involve a Ca2+ signal. Furthermore, as the sensitivities of chloride and iodide effluxes for the anion transporter blockers (especially NPPB) are different, it seems likely that chloride and iodide use two different transport pathways.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Antiporters; Calcimycin; Calcium; Cell Membrane; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Iodides; Kinetics; Nitrobenzoates; Probenecid; Swine; Terpenes; Thapsigargin; Thyroid Gland; Thyrotropin

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Bromides; Cell Communication; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Chlorides; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Iodine; Ion Channels; Membrane Proteins; Nitrates; Nitrobenzoates; Pancreatic Ducts; Rats; Rats, Wistar; Time Factors

Brush-border membrane vesicles were prepared from superficial rat renal cortex by a Mg(2+)-precipitation technique. The initial (20 s) [14C]glucose uptake rate from solutions containing 100 mmol/l Na (salt) was found to be dependent upon the anion composition of the medium; in comparison to gluconate-containing medium (0.46 +/- 0.05 nmol/mg protein), Cl- accelerated the initial rate to 1.47 +/- 0.21 nmol/mg protein (n = 4 preparations, +/- SEM). This enhancement was reduced by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.5 mmol/l), but was unaffected by 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS, 0.5 mmol/l). When membrane vesicles were pre-equilibrated with 100 mmol/l K (salt) and 100 mmol/l mannitol and glucose uptake was measured from a solution containing 100 mmol/l Na gluconate and 100 mmol/l mannitol in the presence of 80 mumol/l valinomycin (to generate an outward K+ diffusion electrical p. d.), it was found that intravesicular KCl depressed the initial glucose uptake compared to K gluconate. NPPB (0.5 mmol/l) increased the initial glucose uptake with intravesicular KCl towards values seen in K gluconate vesicles. In conditions where the only driving force for glucose uptake was established by an inward anion gradient (Nao = Nai) it was found that inward Cl- gradients could drive uphill glucose transport and that this was sensitive to NPPB (0.5 mmol/l), but insensitive to DIDS. We conclude that a Cl- conductance co-exists with Na-cotransport in rat renal brush-border membrane vesicles prepared from superficial renal cortex and this may function to regulate the activity of electrogenic transport systems at this membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chlorides; Glucose; In Vitro Techniques; Ion Channels; Kidney; Male; Membranes; Microvilli; Nitrobenzoates; Potassium Chloride; Rats; Rats, Wistar; Sodium

1. During osmotic swelling, cultured human small intestinal epithelial cells (Intestine 407) exhibited activation of large Cl- currents under the patch-clamp whole-cell configuration. The volume-sensitive Cl- conductance was independent of intracellular Ca2+ and cyclic AMP. 2. The anion permeability sequence of the current was SCN- > I- > Br- > Cl- > F- > gluconate-, corresponding to Eisenman's sequence I. 3. Cl- currents were instantaneously activated by command pulses in a range of -120 to +45 mV. At potentials more positive than +50 mV the current showed a time-dependent inactivation. This inactivation was accelerated by increased depolarization. The instantaneous current-voltage relationship rectified in the outward direction. 4. A stilbene-derivative Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene (SITS), inhibited the Cl- current at micromolar concentrations. SITS facilitated inactivation at positive potentials. Outward currents were more prominently suppressed by SITS than inward currents. The concentrations required for 50% inhibition (IC50) of outward and inward currents were 1.5 and 6 microM, respectively. The outward and inward currents were equally inhibited by a carboxylate analogue Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or diphenylamine-2-carboxylate (DPC) at higher doses (IC50 = 25 for NPPB or 350 microM for DPC). Inactivation kinetics at large depolarizations was not affected by NPPB or DPC. 5. The Cl- current was blocked by an unsaturated fatty acid, arachidonic acid (IC50 = 8 microM). Arachidonic acid was still effective in the presence of inhibitors of lipoxygenase (nordihydroguaiaretic acid, 10 microM), cyclo-oxygenase (indomethacin, 10 microM) and protein kinase C (polymyxin B, 30 microM). The Cl- current was also sensitive to another cis unsaturated fatty acid, oleic acid, which is not a substrate for oxygenases. A trans isomer of oleate, elaidic acid, and a saturated fatty acid, palmitic acid, were ineffective. 6. Single Intestine 407 cells exposed to a hypotonic solution showed a regulatory volume decrease after initial osmotic swelling. The volume regulation was abolished by SITS, NPPB, arachidonate and oleate, but not by elaidate and palmitate. 7. It is concluded that outwardly rectifying Cl- channels, which are sensitive to arachidonic acid, are activated upon osmotic swelling and involved in the subsequent cell volume regulation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Arachidonic Acid; Calcium; Cell Line; Chlorides; Cyclic AMP; Dose-Response Relationship, Drug; Humans; Intestinal Mucosa; Ion Channels; Ion Transport; Membrane Potentials; Nitrobenzoates; Osmotic Pressure

Furosemide has been shown recently to protect asthmatic patients against certain bronchoconstrictor challenges. We investigated the effect of furosemide on eosinophil function. Since furosemide may be exerting its inhibitory effect on the eosinophil by inhibiting anion transport, we also assessed the effects of the anion transport inhibitors 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Furosemide, NPPB and DIDS inhibited the eosinophil respiratory burst in response to leukotriene B4 (LTB4) and, to a smaller extent, inhibited the response to opsonized zymosan (OZ). To assess whether the anion transport inhibitors were achieving their inhibitory effect by inhibiting an influx of Cl- ions into the eosinophil, the effect of removing extracellular Cl- on eosinophil function was determined. OZ-induced H2O2 production was inhibited by removing extracellular Cl- whereas the LTB4 response was not affected by the concentration of extracellular Cl-.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Chlorides; Dose-Response Relationship, Drug; Eosinophils; Furosemide; Guinea Pigs; Hydrogen Peroxide; Leukotriene B4; Nitrobenzoates; Respiratory Burst; Zymosan

The K+ secretory epithelium of the vestibular labyrinth (dark cells) was impaled with glass microelectrodes in order to test the hypothesis that it contains a large Cl- conductance. In the first series of experiments, the short-circuited epithelium was perfused on both sides by a solution containing 150 mmol/l Cl-. The membrane voltage (PD) was -18 +/- 1 mV (N = 101), showed a Gaussian distribution, and the estimated input resistance of the cell (R 'cell') was 17 +/- 3 M omega. The PD responded to 10(-4) mol/l ouabain with a depolarization, suggesting the presence of a (Na(+) + K+)-ATPase. The PD responses to Cl- steps yielded an apparent transference number tCl = 0.34 +/- 0.03 (N = 65) and those to K+ steps yielded a tK = 0.16 +/- 0.01 (N = 48). In the second series of experiments, cells presumed to be Cl(-)-depleted were impaled in Cl(-)-free solutions. The distribution of the PD was not Gaussian; PDs as negative as -90 mV were observed. Cells with a highly negative PD also had a high R 'cell'. With the addition of Cl- the PD collapsed to -19 +/- 1 mV and R collapsed to 16 +/- 3 M omega (N = 145) which are not significantly different from values obtained in the first series of experiments when cells were impaled in a solution containing 150 mmol/l Cl-. Alternating the bath perfusate between Cl(-)-free and Cl(-)-containing solutions led to large PD transients.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Chloride Channels; Chlorides; Diphenylamine; Diuretics; Electrodes, Implanted; Epithelium; Gerbillinae; Membrane Potentials; Membrane Proteins; Nitrobenzoates; Ouabain; Potassium; Sodium-Potassium-Exchanging ATPase; Sulfonamides; Vestibule, Labyrinth

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Calcium; Chloride Channels; Chlorides; Electric Conductivity; Humans; Ion Channels; Ionomycin; Membrane Potentials; Membrane Proteins; Nitrobenzoates; T-Lymphocytes; Zinc; Zinc Compounds

1. A large-conductance Cl- channel was characterized in cell-free membrane patches from the rabbit longitudinal colonic smooth muscle using the patch clamp technique. In addition, the regulation of these channels by neurokinin-1 (NK-1) receptor agonists and G proteins was studied. 2. No spontaneous channel activity was observed in cell-attached patches at the cell resting potential, or in excised patches at pipette potentials (Vp) between -20 and 20 mV. In excised patches, channel activity could be induced in thirty-six out of ninety-six patches by holding the patch at Vp values more negative than -60 mV or more positive than 60 mV. Once induced, the channel showed a bell-shaped voltage activation curve in high symmetric [Cl-], with maximal open probability between 20 and -5 mV. Varying cytosolic calcium concentration ([Ca2+]) between 5 x 10(-8) M and 1.0 mM had no effect on the voltage activation of the channel. 3. In inside-out and outside-out patches, when pipette and bath solutions contained equal [Cl-] (130 mM), the anion channel showed a linear current-voltage (I-V) relationship between -60 and 60 mV with a slope conductance of 309 +/- 20 pS (n = 13). Reversal potential measurements indicated that the channel was selective for Cl- over Na+ and K+ (PCl/PNa = 6:1). 4. Channel openings from the closed state to the full open state as well as transitions through smaller conductance states were observed. The smallest detectable substate had a conductance of 15.6 pS. Based on the similarities in selectivity and linearity of the I-V curve of the smaller conductances with the full open state, and kinetic analysis of channel activity, it is concluded that the large conductance channel is composed of multiple substates which can either open and close independently, or simultaneously via a main gate. 5. The stilbene derivative diiso-thiocyanato-stilbene-disulphonic acid (DIDS) and the diphenylamine-2-carboxylate analogue 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused a dose-dependent, reversible flicker block of the small conductance and significantly reduced the macroscopic current flow through the channel. 6. In quiescent outside-out patches, when the pipette contained a 140 mM-CsCl solution with 10(-6) M-CaCl2, 1.2 mM-MgCl2 and 1 mM-GTP, and the bath contained Ringer solution, addition of the NK-1 receptor antagonists substance P methylester resulted in activation of the full conductance state and of smaller substates.(ABSTRACT TRUNCATED AT 400 WORDS

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chlorides; Colon; Dose-Response Relationship, Drug; Female; GTP-Binding Proteins; Ion Channel Gating; Ion Channels; Male; Membrane Potentials; Muscle, Smooth; Nitrobenzoates; Rabbits; Receptors, Neurokinin-2; Receptors, Neurotransmitter; Substance P

We have investigated the volume-activated transport of organic solutes in flounder erythrocytes. Osmotic swelling of cells suspended in a Na(+)-free medium led to increased membrane transport of taurine, glucose, and uridine. For each compound there was a significant lag period (1-2 min at 10 degrees C) between cell swelling and activation of the flux. The volume-activated fluxes of each of the substrates increased in parallel with increasing cell volume, and those of taurine and uridine increased linearly with concentration (up to 19 mM). The volume-activated fluxes of each of the three compounds showed similar sensitivities to a number of anion-selective channel blockers (5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid approximately MK-196 > niflumic acid > furosemide); the IC50 for the inhibition of the volume-activated fluxes by NPPB was around 12 microM. The results are consistent with the hypothesis that the volume-activated transport of organic osmolytes is via a pathway with the characteristics of a volume-activated "chloride channel." This raises the question of whether the transport of organic substrates might represent a physiological role for such channels in other cell types.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Blood Glucose; Chloride Channels; Diuretics; Erythrocytes; Flounder; Furosemide; Hypotonic Solutions; Indans; Ion Channels; Kinetics; Membrane Proteins; Niflumic Acid; Nitrobenzoates; Taurine; Uridine

The uptakes of gamma-aminobutyrate (GABA) and L-glutamate into synaptic vesicles isolated from rat brain were compared with respect to the effects of 4-acetamido-4'-isothiocyanostilbene-2,2'- disulphonic acid (SITS), 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (N144), agents known to block anion channels. The uptake of glutamate was inhibited by low micromolar concentrations of SITS, DIDS and N144. GABA uptake was much less sensitive to these agents than was glutamate uptake. SITS and N144 inhibited the vacuolar H(+)-ATPase of synaptic vesicles to a smaller extent than the glutamate uptake. The uptake of GABA was not affected by the permeant anions Cl- and Br-, whereas the uptake of glutamate was highly stimulated by low concentrations of these ions. The uptakes of both glutamate and GABA were inhibited by similar, but not identical, concentrations of the lipophilic anion SCN-.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetylcholinesterase; Animals; Anions; Biological Transport; Brain; Ca(2+) Mg(2+)-ATPase; Cell Fractionation; Ethylmaleimide; gamma-Aminobutyric Acid; Glutamates; Kinetics; Male; Neurotransmitter Agents; Nitrobenzoates; Rats; Rats, Inbred Strains; Salts; Synaptic Vesicles; Thiocyanates

The efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from human HCT-8 intestinal epithelial cultured cells was time-dependent, and after 5 hr 76% of the fluorochrome was extracellular. The pharmacological profile for inhibition of this efflux has been investigated, focusing on agents which modulate anion transport. BCECF efflux was sensitive to inhibition by 0.5 mM indomethacin (50% inhibition at 20 microM) which reduced efflux to values observed after depletion of ATP with azide and 2-deoxy-D-glucose. Indomethacin inhibition of BCECF efflux was not reversed with prostaglandin. The stilbene derivatives 4-acetamido-4'-isothiocyano-2-2'-disulphonic stilbene and 4,4'-diisothiocyano-2,2'-disulphonic stilbene only resulted in partial inhibition of BCECF efflux, even at 1 mM. Furosemide, bumetamide, probenecid and 5-nitro-2-(3-phenylpropyl-amino)-benzoate only reduced BCECF efflux at 1 mM. The cationic agent vinblastine was as active as indomethacin as an inhibitor of BCECF efflux (50% inhibition) with 10 microM) while actinomycin D was also a good inhibitor (50% inhibition with 100 microM). Several other cationic agents, including nifedipine, amiloride and reserpine, were ineffective as inhibitors of BCECF efflux in concentrations up to 1 mM. Thus, the pharmacological profile for inhibition of BCECF efflux does not fully equate with any recognised transport system. Agents such as cytochalasin B and chloroquine did not fully equate with any recognised transport system. Agents such as cytochalasin B and chloroquine did not effect BCECF efflux suggesting accumulation and subsequent discharge from endosomes is not a pathway for secretion. BCECF may be a substrate for a cellular secretory detoxifying system in epithelial cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Biological Transport; Dose-Response Relationship, Drug; Epithelium; Fluoresceins; Humans; Hydrogen-Ion Concentration; Indomethacin; Intestinal Neoplasms; Methotrexate; Nitrobenzoates; Spectrometry, Fluorescence; Tumor Cells, Cultured; Vinblastine

We microperfused the loop of Henle (LOH) to assess its contribution to urine acidification in vivo. Under control conditions (Na HCO3- = 13 mM, perfusion rate approximately 17 nl/min-1) net bicarbonate transport (JHCO3-) was unsaturated, flow- and concentration-dependent, and increased linearly until a bicarbonate load of 1,400 pmol.min-1 was reached. Methazolamide (2 x 10(-4) M) reduced JHCO3 by 70%; the amiloride analogue ethylisopropylamiloride (EIPA) (2 x 10(-4) M) reduced JHCO3 by 40%; neither methazolamide nor EIPA affected net water flux (Jv). The H(+)-ATPase inhibitor bafilomycin A1 (10(-5) M) reduced JHCO3 by 20%; the Cl- channel inhibitor 5-nitro-2'-(3-phenylpropylamino)-benzoate (2 x 10(-4) M) and the Cl(-)-base exchange inhibitor diisothiocyanato-2,2'-stilbenedisulfonate (5 x 10(-5) M), had no effect on fractional bicarbonate reabsorption. Bumetanide (10(-6) M) stimulated bicarbonate transport (net and fractional JHCO3-) by 20%, whereas furosemide (10(-4) M) had no effect on bicarbonate reabsorption; both diuretics reduced Jv. In summary: (a) the LOH contributes significantly to urine acidification. It normally reabsorbs an amount equivalent to 15% of filtered bicarbonate; (b) bicarbonate reabsorption is not saturated; (c) Na(+)-H+ exchange and an ATP-dependent proton pump are largely responsible for the bulk of LOH bicarbonate transport.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Absorption; Acid-Base Equilibrium; Amiloride; Animals; Anti-Bacterial Agents; Bicarbonates; Biological Transport; Bumetanide; Furosemide; Loop of Henle; Macrolides; Male; Methazolamide; Nitrobenzoates; Perfusion; Permeability; Rats; Rats, Inbred Strains

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is characterized by its high content of Na+/K(+)-ATPase and a Cl- conductance, which function in parallel in salt reabsorption. In order to reconstitute the Cl- channels, TALH membrane vesicles were solubilized in 1% sodium cholate in buffer containing 200 mM KCl, followed by dilution with soybean lipids (final ratio of protein/detergent/lipid of 1:3:15 in mg) and removal of the detergent by gel filtration on Sephadex G-50. Cl- channel activity in the liposomes was determined by a 36Cl- uptake assay where the accumulation of the radioactive tracer against its chemical gradient is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The 36Cl- uptake by the KCl-loaded liposomes was dependent on the inclusion of membrane protein and was abolished by valinomycin, indicating the involvement of a conductive pathway. It was also inhibited by 36% by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Solubilization of the Cl- channels in cholate was optimal in the presence of 200 mm KCl, but was found to decrease markedly at low ionic strength. SDS-PAGE analysis of the proteins extracted by cholate at high and low salt concentrations showed that the Cl- channel-containing high KCl extract was enriched in the 96 and 55 kDa alpha- and beta-subunits of the Na+/K(+)-ATPase (the major proteins in the membrane preparation) and several minor protein bands. Treatment of the membrane vesicles with the radioactive analogue of DIDS, [3H]2DIDS, labeled primarily a 65 and a 31 kDa protein. The solubilization of the 31 kDa protein by cholate depended markedly on the ionic strength and thus paralleled the solubilization pattern of Cl- channel activity. Furthermore, the labeling of the 31 kDa protein was prevented by nonradioactive DIDS and by NPPB but not by other compounds, indicating that it may be a Cl- channel component.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cell Membrane; Chloride Channels; Chlorides; Chlorine; Chromatography, Gel; Kidney Tubules; Liposomes; Loop of Henle; Membrane Potentials; Membrane Proteins; Nitrobenzoates; Potassium Chloride; Radioisotopes; Sodium-Potassium-Exchanging ATPase; Solubility; Swine; Valinomycin

Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl- channels with a single-channel conductance of 73 +/- 2 pS in symmetrical 140 mM KCl solution (n = 25). The current-voltage relationship was linear in the range of -60 to +80 mV, but channel kinetic properties depended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10-15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2-6 msec. Clamp voltages more negative than -50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl- = Br- = I- greater than SO4(2-) approximately F-. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1.0 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1 mM) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl(-)-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chloride Channels; Chlorides; In Vitro Techniques; Ion Channels; Kidney Cortex; Membrane Fusion; Membrane Potentials; Membrane Proteins; Nitrobenzoates; Organelles; Rats; Rats, Inbred Strains

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

Incubation of human skin fibroblasts in hypotonic media induced the activation of 36Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of 36Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also 36Cl- influx was enhanced by hypotonic medium.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Biological Transport; Chloride Channels; Chlorides; Chlorine; Fibroblasts; Furosemide; Humans; Hypotonic Solutions; Kinetics; Membrane Proteins; Nitrobenzoates; Osmolar Concentration; Osmotic Pressure; Radioisotopes

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber in order to identify and localize chloride conductances. The [Cl-] was rapidly reduced (substituted with isethionate) in the apical or basolateral perfusate and the transepithelial potential difference (Vt) and transepithelial resistance (Rt) were monitored continuously. In addition, agents known to inhibit anion transport in other epithelia were applied. The direction of all initial changes in Vt and Rt due to Cl- substitutions were consistent with the presence of ionic conductances for Cl- on both sides of the epithelium. The time-courses and magnitudes of the fall in Vt and increase in Rt during apical [Cl-] steps in the presence and absence of basolateral bumetanide were monophasic and identical in the two cases. The response of Vt to basolateral [Cl-] steps was biphasic and the initial response was greatly attenuated by bumetanide. These findings demonstrate that the largest conductance for Cl- is in the basolateral cell membrane, but that the paracellular and/or apical pathway also possess a finite Cl- conductance. All three agents tested, 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), caused an increase in Vt. NPPB and DIDS were more effective from the apical side. DCDPC and DIDS administered from the apical side led to a decrease in Rt. These results suggest that these agents act in this tissue by enhancing a conductive pathway on the apical membrane rather than blocking the basolateral Cl- conductance.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bumetanide; Chloride Channels; Chlorides; Diphenylamine; Electric Conductivity; Epithelium; Gerbillinae; Kinetics; Membrane Potentials; Membrane Proteins; Nitrobenzoates; Saccule and Utricle