4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and fenamic-acid

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

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

Assessment of functional EP receptor subtypes involved in PGE2-induced secretion in human duodenum. The spectrum of activities by PGE2 in mammals, including cytoprotective bicarbonate secretion in duodenum, is mediated through four G protein-coupled receptor subtypes (EP1-EP4).. Biopsies from the second part of duodenum from patients undergoing endoscopy were mounted in modified Ussing chambers. Basal and stimulated short circuit current (SCC) and slope conductance (SG) were measured. Dose-response relations for PGE2 and subtype receptors EP1/EP3 (sulprostone), EP2 (butaprost), and EP4 (1-OH PGE1) were assessed by cumulated doses of single agonists.. PGE2 caused a dose-dependent increase in SCC, maximum 101 +/- 20 microA cm(-2) with an EC50 of 35.6 +/- 5.8 nm (n = 3). Likewise 1-OH PGE1 caused a dose-dependent SCC increase, maximum 63.3 +/- 28.6 microA cm(-2) with an EC50 of 56.7 +/- 7.2 nm (n = 3). 1-OH PGE1 at 500 nm increased SCC by 18.0 +/- 3.0 microA cm(-2) (n = 10) and SG by 2.9 +/- 0.4 mS cm(-2) (n = 6). Sulprostone (n = 6) and butaprost (n = 6) had no effects on SCC or SG. SCC was inhibited 31.4 +/- 13.2% (n = 10) by bumetanide (25 microM serosa) and 18.6 +/- 5.8% (n = 10) by acetazolamide (250 microM lumen). Diphenylamine-2-carboxylate (DPC) (250 microM mucosa) and SITS (10 microM mucosa) had almost no effect.. Effects of PGE2 on secretion in the second part of human duodenum is mediated through the EP4 receptor and not through EP1, EP2, or EP3.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Alprostadil; Bicarbonates; Chlorides; Dinoprostone; Dose-Response Relationship, Drug; Duodenum; Humans; ortho-Aminobenzoates; Prostaglandins; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP4 Subtype

Acetylcholine (ACh) stimulates ion secretion in the small intestine and colon. The purpose of the present study was to characterize the ACh-induced electrogenic ion transport in human duodenum and determine the muscarinic receptor subtypes functionally involved.. Biopsies from the second part of duodenum were obtained from 28 patients during endoscopy. Biopsies were mounted in modified Ussing chambers with air-suction for measurements of short-circuit current by a previously validated technique. Short-circuit current was measured after application of chloride/bicarbonate transport inhibitors bumetanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), diphenylamine-2-carboxylate (DPC), and acetazolamide. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and two mamba toxins MT3 and MT7 were used to characterize the mAChR receptor subtypes involved. The effects of transport inhibitors and receptor antagonists were measured by comparing two consecutive responses of ACh on short-circuit current in the same biopsy specimen.. Bumetanide and 4-DAMP significantly inhibited ACh-induced short-circuit current, whereas SITS, DPC, acetazolamide, mamba toxin MT3, and mamba toxin MT7 all failed to show any significant effect.. In conclusion, our results indicate that muscarinic receptor subtype M3 acts as the main mediator of bumetanide-sensitive ACh-induced secretion in human duodenum.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Acetylcholine; Bumetanide; Calcium Channel Blockers; Diuretics; Duodenum; Elapid Venoms; Humans; Intercellular Signaling Peptides and Proteins; Ion Transport; Muscarinic Antagonists; Neurotoxins; ortho-Aminobenzoates; Peptides; Piperidines; Receptors, Muscarinic

Rat distal colon epithelium is frequently employed to assess the effect of natural and synthetic chemicals on chloride secretion. Inhibition of chloride secretion is often reported as the loop diuretic-sensitive portion of short-circuit current (Isc). The present work challenges the hypothesis that a loop diuretic alone is able to fully abolish chloride secretion. Isolated mucosa preparations were mounted in an Ussing chamber. The effects on short-circuit current of replacement of normal Ringer by a low (2.5 mmol/L) Cl solution and of blockers of basolateral Na, K, 2 Cl symport (bumetanide), apical Cl channels (diphenylamine-2-carboxylate, DPC), and anion exchange (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, SITS) alone and combined were assessed. Low Cl reversibly decreased Isc by 76%. In normal Ringer, bumetanide decreased Isc by 65%. SITS also had a significant effect at the serosal side, but not at the apical side, where DPC caused a 40% decrease. Chloride replacement, bumetanide and DPC, but not SITS, increased epithelial resistivity. Combined blockade of Na, K, 2 Cl symport and apical Cl channels, of Na, K, 2 Cl symport and anion antiport, or of anion antiport and apical Cl channels was needed to achieve reduction of short circuit current to the same extent seen with chloride replacement. Present results indicate that Isc of the unstimulated epithelium is mostly due to chloride secretion, and at least two blockers are required to abolish it. This fact should be taken into account in studies of chloride secretion-stimulating agents.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Bumetanide; Calcium Channel Blockers; Chlorides; Colon; Diuretics; Dose-Response Relationship, Drug; Electric Conductivity; Intestinal Mucosa; Male; ortho-Aminobenzoates; Rats; Rats, Wistar

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 lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+-ATPase [proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the corpus and cauda epididymis and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase type II (CAII) and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 +/- 6.1%. The rate of acidification was independent of Cl- and was strongly inhibited by SITS under both normal and Cl--free conditions (50.6 +/- 5.0 and 57. 5 +/- 6.0%, respectively). In the presence of Cl-, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 +/- 11.3% when applied together with DPC. In Cl--free solution, DPC markedly inhibited proton efflux by 45.1 +/- 7.6%, SITS produced an additional inhibition of 18.2 +/- 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of Cl-, but DPC-sensitive Cl- channels might contribute to basolateral extrusion of HCO-3 under Cl--free conditions. The inhibition by SITS observed under both normal and Cl--free conditions indicates that a Cl-/HCO-3 exchanger is not involved and that an alternative HCO-3 transporter participates in proton secretion in the proximal vas deferens.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Animals; Anti-Bacterial Agents; Bicarbonates; Biological Transport; Carbonic Anhydrases; Chlorides; Enzyme Inhibitors; Hydrogen-Ion Concentration; Kinetics; Macrolides; Male; ortho-Aminobenzoates; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Vacuoles; Vas Deferens

We tested the hypothesis that the early action potential shortening induced by hypoxia in perfused hearts is attributable to chloride currents activated or modulated by endogenous catecholamine release. Rabbit hearts perfused at 33 degrees C and paced at 2.5-2.8 Hz were used for membrane potential recordings with microelectrodes. Catecholamine depletion was induced with reserpine treatment. The effects of nadolol (10 microM), the stilbenedisulfonic acid derivatives DIDS (10 microM) and SITS (1 mM), and diphenylamine-2 carboxylate (DPC, 100 microM) on action potential characteristics were determined at different times during hypoxia. The effect of chloride transport blockers on the outward currents induced by 200 nM carbonyl cyanide (CCCP) or by 1 microM isoproterenol in isolated cells was also tested. In control hearts, action potential duration (APD) at 25 and 95% repolarization decreased by 50 +/- 9% and 32 +/- 7% respectively after 5 min of hypoxia. This effect was fully antagonized by reserpine pretreatment, by respiratory acidosis, and by nadolol when present from the beginning of hypoxia. None of these agents affected action potential characteristics in normoxia and nadolol had no effect when added after 15 min of hypoxia. Lowering the chloride concentration to 17.5 mM reproduced the effects of nadolol and reserpine. DIDS and SITS lengthened APD in normoxia and attenuated the early APD shortening in hypoxia. DPC had no effect in normoxia but fully counteracted APD shortening produced by isoproterenol or early hypoxia. In isolated cells, DIDS did not affect the glibenclamide sensitive outward current induced by CCCP and DPC blocked the isoproterenol induced current. The data suggest that in whole hearts, chloride currents mediated by endogenous catecholamine release are involved in the early action potential shortening induced by hypoxia with preservation of glycolysis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Action Potentials; Adrenergic Agonists; Animals; Catecholamines; Cell Hypoxia; Chloride Channels; Heart Ventricles; In Vitro Techniques; Isoproterenol; ortho-Aminobenzoates; Rabbits; Ventricular Function

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl- channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately -15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl-. Readdition of Ca2+ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca2+ inactivated Cl- channel (CaIC). The inhibitory potency of Ca2+ was a function of the external Ca2+ concentration with a half maximal blocker concentration of about 20 microM. These channels were inhibited by the Cl- channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4'-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonicacid (SITS), another Cl- channel blocker, led to activation of this Cl- channel. Like other Cl- channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl- current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton. The results indicate that removal of divalent cations activates Cl- channels in Xenopus oocytes which share several features with Cl- channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl- channels.

Topics: 1-Methyl-3-isobutylxanthine; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adenosine Triphosphate; Animals; Calcium; Cell Membrane Permeability; Chloride Channels; Cholera Toxin; Cyclic AMP; Cytochalasin D; Cytoskeleton; Flufenamic Acid; Membrane Potentials; Oocytes; Oogenesis; ortho-Aminobenzoates; Patch-Clamp Techniques; Pertussis Toxin; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella; Xenopus laevis

In vivo studies of fetal sheep suggest that the liquid present in the lumen of the lung throughout fetal life is derived from Cl- secretion by the pulmonary epithelium. Monolayer preparations of enriched epithelial cells from distal fetal rat (18-day gestation) lung, grown in serum-free media, were histologically similar to acinar (prealveolar) structures of fresh tissue. In Ussing chambers, basal transepithelial potential difference (PD), calculated equivalent short-circuit current (Ieq), and transepithelial resistance (R) were 4.4 +/- 0.3 mV (matrix positive), 35.6 +/- 1.6 microA/cm2, and 120.0 +/- 4.0 omega cm2, respectively. Ouabain (10(-3) M) eliminated 57% of basal Ieq within 30 min, amiloride (10(-4) M) induced a 13% fall in Ieq, and phlorizin (10(-4) M) had no effect on bioelectric properties. Diphenylamine-2-carboxylate (DPC, 3 x 10(-3) M) inhibited Ieq by 50%. Bumetanide had no effect on baseline bioelectric parameters. The hyperpolarization that accompanied apical or bilateral replacement of Cl- and was enhanced by terbutaline suggested an apical Cl- permselectivity. Effects of Na+ replacement on amiloride-pretreated monolayers were consistent with Na(+)-dependent Cl- secretion or amiloride-insensitive pathways. Under these growth conditions, this preparation exhibits bioelectric characteristics that are compatible with Cl- secretion and Na+ absorption. The mechanism of Cl- secretion may be similar to that of airways but is uniquely bumetanide insensitive.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Cells, Cultured; Chlorides; Electrophysiology; Fetus; Lung; Microscopy, Electron; ortho-Aminobenzoates; Ouabain; Rats; Sodium; Terbutaline

Studies were performed in strips of opossum lower esophageal sphincter (LES) muscle in vitro. External Cl(-)-free Krebs solution (0[Cl-]o) inhibited resting tone. Treatment with the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 0.3-100 microM) caused a concentration-dependent relaxation of LES muscle, as did treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1 microM-3 mM), a Cl(-)-HCO3- exchange blocker, and bumetanide (0.3-100 microM), a blocker of the Na(+)-K(+)-2Cl- cotransport. DIDS and bumetanide are also known to cause Cl- channel block. The calculated pD2 and Emax values for DPC, DIDS, and bumetanide were 5.24 +/- 0.28 (n = 5), 3.38 +/- 0.16 (n = 5), and 4.49 +/- 0.23 M (n = 5), and 78.80 +/- 5.38, 74.80 +/- 6.54, and 83.70 +/- 10.20%, respectively. The neuronal Cl- channel activators gamma-aminobutyric acid and glycine had no effect on the resting tone. DPC, DIDS, and bumetanide appear to have acted directly on smooth muscle rather than indirectly through the release of inhibitory neurotransmitters because LES relaxation by these agents was not influenced by tetrodotoxin (10 microM), which blocks action potentials in nerves, or by omega-conotoxin (1 microM), which inhibits the release of neurotransmitters from nerve terminals. LES muscle relaxed by exposure to 0[Cl-]o, DPC, DIDS, and bumetanide contracted with the addition of carbachol (30 microM); muscle so treated was resistant to the inhibitory neurotransmitter-mediated relaxation ordinarily induced by electrical field stimulation (EFS, 0.12-32 Hz). This effect was not nonselective, as the EFS-resistant muscle relaxed fully with isoproterenol (0.1-100 microM). HCO(3-)-free Krebs in the nominal absence of CO2 did not affect the resting tone and its relaxation. The Ca2+ channel blocker nifedipine decreased resting tone but did not antagonize the relaxation of carbachol-contracted muscle induced by either EFS or isoproterenol. These studies suggest that Cl- may play an important role in LES tone and relaxation due to inhibitory neurotransmitter released from intramural nerves.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bumetanide; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Esophagogastric Junction; Female; Ions; Male; Muscle Relaxation; Muscle Tonus; Nifedipine; Opossums; ortho-Aminobenzoates; Osmolar Concentration

We examined the effects of formate and oxalate on the rate of fluid absorption (Jv) in the rat proximal convoluted tubule in situ. Proximal tubules were microperfused with a high-Cl-, low-HCO3- Ringer solution (pH 6.7), and the peritubular capillaries were perfused with a standard Ringer solution (pH 7.4), simulating conditions in the late proximal tubule. Jv, a measure of transtubular NaCl absorption under these conditions, was calculated from the change in luminal [3H]inulin. Addition of formate in the physiological range (500 microM) to the luminal perfusate increased Jv by 45%; addition of 500 microM formate to both luminal and capillary perfusates increased Jv by 57%. Similarly, addition of oxalate in the physiological range (5 microM) to the luminal perfusate increased Jv by 37%; addition of 5 microM oxalate to both luminal and capillary perfusates increased Jv by 57%. The stimulatory effects of formate and oxalate perfused in the lumen and capillaries were not additive. Addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, 0.1 mM) to the luminal perfusate had no effect on baseline Jv measured in the absence of added formate and oxalate but completely abolished the increment in Jv induced by formate and oxalate. Addition of the Cl(-)-channel blocker diphenylamine-2-carboxylate (DPC, 0.2 mM) to the capillary perfusate had no effect on baseline Jv but completely abolished the increment in Jv induced by formate and oxalate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Absorption; Animals; Chlorides; Formates; Ion Exchange; Kidney Tubules, Proximal; Male; ortho-Aminobenzoates; Oxalates; Perfusion; Rats; Rats, Inbred Strains; Sodium Chloride

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bicarbonates; Buffers; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Fluoresceins; Histamine; Hydrogen-Ion Concentration; Kinetics; ortho-Aminobenzoates; Parietal Cells, Gastric; Quinolinium Compounds; Rabbits; Spectrometry, Fluorescence

The hypothesis was tested that the uterus of the rat orally infected with the parasite Trichinella spiralis becomes hypersensitized and that subsequent antigenic challenge affects functions in the endometrial epithelium. Results of experiments comparing the immunological responsiveness of isolated rat uterus with that of the jejunum supports our hypothesis. Antigenic challenge of uterus mounted in Ussing-type chambers causes an elevation in transuterine short circuit current (Isc) of 6.4 +/- 0.8 microA/cm2. The transduction of the antigenic signal to elicit the electrophysiological response involves 5-hydroxytryptamine (5-HT) working through a nerve-independent pathway. The antigen-stimulated rise in Isc peaks approximately 3 min after challenge. The uterine response is blocked by diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of bicarbonate-chloride exchange. The antigen-evoked change in jejunal Isc is biphasic, peaking at 1.5 and approximately 4.0 min after challenge, and is about 10-fold greater in magnitude than the Isc in the uterus. The transductive pathway in the jejunum involves 5-HT, histamine and prostaglandin acting partly through intrinsic nerves. The jejunal response to antigen is inhibited by diphenylamine-2-carboxylate, a chloride channel blocker. Changes in net ion transport which are primed by infection and evoked by antigen are apparently triggered by local anaphylaxis in both the uterus and jejunum.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Analysis of Variance; Animals; Antigens, Helminth; Biological Transport; Carbachol; Cinanserin; Dinoprostone; Endometrium; Female; Histamine; In Vitro Techniques; Indomethacin; Jejunum; ortho-Aminobenzoates; Ovalbumin; Rats; Rats, Inbred Strains; Regression Analysis; Serotonin; Signal Transduction; Trichinella

Compounds that prevent chloride transport in membrane vesicles have been tested for in vivo activity against the effects of intestinal secretory agents. Chloride channel blockers including diphenylamine-2-carboxylate, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate, 5-nitro-2-(2-phenylethylamino)benzoic acid, and alpha-phenylcinnamic acid were tested for effects on jejunal or ileal secretion in weanling pigs. Secretion was studied in ligated intestinal loops in a control state, during exposure to secretory concentrations of theophylline, and after prior treatment with cholera toxin. Increases in net fluid flux induced by either theophylline or cholera toxin were not prevented by adding chloride channel blockers into the intestinal lumen. Channel blocker concentrations that reduced chloride transport by greater than 50% in pig jejunal brush border vesicles did not cause significant changes in unidirectional blood to lumen chloride flux measured in situ. Several routes of administration of the specific chloride channel blocker alpha-phenylcinnamate failed to reduce fluid secretion induced by theophylline. Chloride channel blocker effectiveness appears to be significantly different between in vitro and in vivo experimental models. In contrast to the chloride channel blockers, loperamide significantly reduced net fluid and chloride flux in ileal loops secreting fluid in response to theophylline. Antagonism of the production or actions of second messenger by loperamide was more effective than the chloride channel blockers in reducing conductive chloride transport associated with intestinal secretion.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Chloride Channels; Cholera Toxin; Cinnamates; Ileum; Jejunum; Ligation; Loperamide; Membrane Proteins; ortho-Aminobenzoates; Swine; Theophylline

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Benzothiazoles; Carbocyanines; Chlorides; Fluorescent Dyes; Ion Channels; Kidney Medulla; Membrane Potentials; Membranes; ortho-Aminobenzoates; Swine; Valinomycin

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

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Calcium; Chloride Channels; Chlorides; Membrane Proteins; Microinjections; Oocytes; ortho-Aminobenzoates; Pituitary Gland, Anterior; Pituitary Hormone-Releasing Hormones; Receptors, Gonadotropin; RNA, Messenger; Xenopus laevis

Intracellular pH (pHi) of primary monolayer cultures of the rat epididymal cells has been measured using fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). When incubated in normal Krebs-Henseleit (K-H) solution containing 25 mM HCO3, cell monolayers exhibited a basal pHi of 7.17 +/- 0.08. Amiloride (0.5 mM) added to the cell monolayers caused an immediate and sustained fall in pHi by 0.43 +/- 0.05 pH unit. Similar effects were produced when extracellular Na ions were substituted by N-methyl-D-glucamine. Addition of SITS (0.5 mM) or DPC (0.6 mM) resulted in a transient intracellular alkalosis. Adrenaline (0.23 microM) caused a fall in pHi by 0.33 +/- 0.05 pH unit. The fall was slow and was achieved after 30 min (half time 9.9 min). The effect of adrenaline was reversible upon washing and was blocked by propranolol (2 microM). The effect of adrenaline was mimicked by forskolin (10 microM) and Br-cAMP (0.5 mM) which caused a fall in pHi by 0.35 +/- 0.03 and 0.35 +/- 0.02 pH unit respectively. Addition of diphenylamine-2-carboxylate (DPC, 0.6 mM) to the cuvette completely blocked the intracellular acidification produced by adrenaline or forskolin. However, addition of SITS (0.5 mM) did not prevent the intracellular acidosis induced by these agonists. If monolayers were first treated with forskolin (10 microM), the intracellular pH fell, when stabilized, subsequent addition of amiloride caused a further fall in pHi. When incubated in a Cl-free solution (Cl substituted by gluconate), cell monolayers exhibited a pHi of 7.23 +/- 0.07 (values not significantly different from monolayers incubated in HCO3 solution).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Cells, Cultured; Epididymis; Epinephrine; Hydrogen-Ion Concentration; Male; ortho-Aminobenzoates; Rats; Rats, Inbred Strains

Chloride transport across human placental microvillous vesicle membrane was investigated using the fluorescent probe SPQ (6-methoxy-N[3-sulfopropyl]quinolinium). Chloride influx (JCl) was calculated from the initial rate of quenching of intravesicular SPQ fluorescence by chloride. JCl measured by SPQ fluorescence was not significantly different from JCl measured by uptake of 36Cl; SPQ did not affect measurements of JCl.JCl was increased 51% by a 58-mV membrane potential (internal positive). Voltage-stimulated JCl showed a saturable dependence on chloride concentration with a dissociation constant (Kd) of 18 +/- 5 mM and was inhibited by diphenylamine-2-carboxylate with an apparent inhibitory constant of 0.13 +/- 0.03 mM. The activation energy calculated for voltage-stimulated JCl was 4.6 +/- 0.6 kcal/mol. JCl was also stimulated by a reduction in the external pH from 7.0 to 5.5 (internal pH = 7.0). pH-stimulated chloride influx was increased by trans-HCO3 (25 mM) and was inhibited by dihydro-4,4'-diisothiocyano-2,2'-disulfonic stilbene. Uptake of 36Cl into microvillous vesicles was stimulated by trans-Cl. pH-stimulated JCl showed a saturable dependence on chloride with a Kd of 38 +/- 6 mM but was not affected by membrane potential. No evidence was found for Na- or K-coupled chloride cotransport. These findings demonstrate the presence of a saturable chloride conductance and an electroneutral chloride-bicarbonate exchanger in the placental microvillous membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Bicarbonates; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chlorides; Female; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Kinetics; Mathematics; Microvilli; Models, Theoretical; ortho-Aminobenzoates; Placenta; Potassium; Pregnancy; Quinolinium Compounds; Spectrometry, Fluorescence; Valinomycin

The patch-clamp method was applied to the lateral membrane of late proximal tubules of the rabbit kidney. Tubule segments were cannulated on one side by a perfusion system. At the noncannulated end of the tubules, the lateral membrane was accessible to a patch pipette. In cell-attached, as well as cell-excised (presumably inside-out oriented) membrane patches, a voltage sensitive channel was observed. The open-state probability of this channel increased with depolarizing potentials. In cell-excised patches bathed with NaCl-Ringer on both sides, the single channel conductance g was 28.0 +/- 1.2 pS (n = 10). With KCl-Ringer in the pipette and NaCl-Ringer in the bath g was 24.7 +/- 1.3 pS (n = 7) and the current-voltage curve crossed the axis at 0 mV. Therefore, the channel does not discriminate between K+ and Na+ ions. Replacing half of NaCl by mannitol on the bath side yielded a permeability for cations about twice as high as for Cl-. The channel could be reversibly blocked by diphenylamine-2-carboxylate (DPC), whereas its inhibition by SITS was only partially reversible. In cell-attached patches, the channel was nearly inactivated at zero clamp potential, but became active when the membrane patch was depolarized. The significance of this nonselective channel for proximal tubule cell function is still unclear. It could be involved in the contraluminal exit mechanism of various anions. However, it could also play a role in cell volume regulation processes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Basement Membrane; Cell Membrane Permeability; Chlorides; In Vitro Techniques; Ion Channels; Kidney Tubules, Proximal; Membrane Potentials; ortho-Aminobenzoates; Potassium; Rabbits; Sodium

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide-sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anthracenes; Biological Transport, Active; Bromides; Bucladesine; Bumetanide; Calcimycin; Cell Line; Cell Membrane; Chlorides; Colon; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Humans; Hydrogen-Ion Concentration; Kinetics; ortho-Aminobenzoates; Sodium; Valinomycin; Vasoactive Intestinal Peptide