nystatin-a1 and fenamic-acid

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.

Topics: 1-Methyl-3-isobutylxanthine; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cell Polarity; Chloride Channels; Chloride-Bicarbonate Antiporters; Chlorides; Colforsin; Epithelial Cells; Female; In Vitro Techniques; Lung; Membrane Potentials; Nitrobenzoates; Nystatin; ortho-Aminobenzoates; Permeability; Time Factors; Xenopus laevis

The DeltaF508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and "chemical chaperones" allow DeltaF508-CFTR to traffic to the plasma membrane and increase Cl(-) permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce DeltaF508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing DeltaF508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl(-) secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl(-) current across the apical plasma membrane. Thus butyrate increased the amount of functional DeltaF508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl(-) secretion because of inhibitory effects on Cl(-) uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on DeltaF508-CFTR will allow investigation of DeltaF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.

Topics: Animals; Butyrates; Calcium Channel Blockers; Cell Line; Cell Membrane; Cell Polarity; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Enzyme Inhibitors; Epithelial Cells; Genistein; Green Fluorescent Proteins; Humans; Indicators and Reagents; Ionophores; Luminescent Proteins; Microscopy, Confocal; Nystatin; ortho-Aminobenzoates; Protein Transport; Recombinant Fusion Proteins; Temperature; Transgenes

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

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

We studied the effect of tetracyclines on the Na+/K+ pump activity in Calu-3, a human airway cell line. To estimate Na+/K+ pump capacity on the basolateral membrane, an ouabain-sensitive component of the short-circuit current (Isc) was measured in the presence of nystatin, an ionophore of Na+. The application of ouabain (1 mM) to the basolateral solution completely inhibited the Isc generated by adding nystatin (50 microM) to the apical solution. Tetracycline (TC), minocycline (MC), or demethylchlortetracycline (DC) at 0.5 mM applied to the apical but not to the basolateral solution also decreased the nystatin-induced Isc. Neither phlorizin- nor diphenylamine-2-carboxylic acid-sensitive Isc was affected by TC, MC, or DC. These results indicate that tetracyclines may permeate only through the apical membrane with the result that the Na+/K+ pump's capacity for Na+ extrusion should be suppressed without a decrease in Cl- transport.

Topics: Anti-Bacterial Agents; Bronchi; Calcium Channel Blockers; Cell Line; Demeclocycline; Electrophysiology; Enzyme Inhibitors; Humans; Hydrochloric Acid; Ionophores; Minocycline; Nystatin; ortho-Aminobenzoates; Ouabain; Phlorhizin; Sodium-Potassium-Exchanging ATPase; Time Factors