Activation of CFTR Cl(-) channel by tyrphostins via a protein tyrosine kinase-independent pathway in forskolin-stimulated renal epithelial A6 cells

We studied effects of tyrphostin A23 (an inhibitor of protein tyrosine kinase; PTK) and tyrphostin A63 (an inactive analog of tyrphostin A23) on forskolin-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and Cl(-) secretion in renal epithelial A6 cells. Tyrphostin A23 and A63 had no effects on the basal CFTR Cl(-) channel and Cl(-) secretion. However, under the forskolin-stimulated condition, tyrphostin A23 and A63 stimulated Cl(-) secretion by activating CFTR Cl(-) channels. These observations suggest that: 1) tyrphostin A23 and A63 stimulate the cAMP-activated CFTR Cl(-) channel via a PTK-independent, structure-dependent mechanism, and 2) tyrphostin A23 and A63 do not stimulate the basal CFTR Cl(-) channel. These lead us to an idea that: 1) cAMP might cause a conformational change of CFTR Cl(-) channel which is accessible by tyrphostins, and 2) tyrphostins would stimulate translocation of the cAMP-modified channel to the apical membrane by binding to the channel.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Airway epithelial cells-Functional links between CFTR and anoctamin dependent Cl(-) secretion

Airways consist of a heterogeneous population of cells, comprising ciliated cells, Clara cells and goblet cells. Electrolyte secretion by the airways is necessary to produce the airway surface liquid that allows for mucociliary clearance of the lungs. Secretion is driven by opening of Cl(-) selective ion channels in the apical membrane of airway epithelial cells, through either receptor mediated increase in intracellular cAMP or cytosolic Ca(2+). Traditionally cAMP-dependent and Ca(2+)-dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1) and with detailed knowledge of the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR), it has become possible to look more closely into this relationship.     

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells

Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO₃across the tracheal epithelium. The mechanism of the defect in HCO₃ secretion is ill defined; however, a lack ofapical Cl/HCO₃ exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO₃ exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO₃exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO₃ exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO₃ exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO₃ exchanger activity intracheal cells. We propose that the tracheal HCO₃secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO₃exchange activity mediated by DRA.

     

Expression of CFTR and Cl(-) conductances in cells of pulmonary neuroepithelial bodies

The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl(-) conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease.     

Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel

The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels remain controversial. In this work, the contributions made by ATP and Mg(2+) ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg(2+)] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg(2+)-dependent step that followed binding of both ATP and Mg(2+). Once a channel had opened, sudden withdrawal of all Mg(2+) and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg(2+) ions alone could close it, and the closing rate increased with [Mg(2+)] over the micromolar range (half maximal at approximately 50 microM [Mg(2+)]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg(2+) ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.     

Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

CFTR targeting in epithelial cells

We used polarized and nonpolarized colonic cell lines (HT-29) to correlate CFTR function and expression with epithelial cell morphogenesis. Unpolarized cells express levels of CFTR mRNA and protein that are equivalent to those observed in polarized cells, and the extent of CFTR glycosylation is also similar. Despite these similarities in CFTR expression, the polarized cells secreted Cl in response tocAMP, but there was nocAMP-stimulated Cl conductance response in the unpolarized cells. In the polarized cells, CFTR is localized in the apical membrane domain, but in unpolarized cells the protein is retained at a perinuclear location. These findings indicate that a peripheral targeting mechanism, distal to the Golgi cisternae, controls the progression of N-linked glycoproteins like CFTR to the apical membrane. This targeting process does not become active until epithelial cells polarize. It may determine whether mutant forms of CFTR are targeted to the apical membrane.     

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.     

cAMP-dependent Cl(-) channel protein (CFTR) and its mRNA are expressed in the secretory portion of human eccrine sweat gland.

Immunoreactive cystic fibrosis transport regulator (CFTR) proteins in human sweat ducts has been documented but CFTR expression in the secretory coil has remained uncertain. Using monoclonal antibodies (MAbs) against epitopes in the R-domain and C-terminus, we observed the following: Formalin fixation masks the CFTR epitopes but the epitopes are exposed by treatment with urea and heat (antigen retrieval). Pen-Fix fixation preserves CFTR epitopes. The secretory coil also expresses CFTR epitopes for the R-domain and C-terminus. An MAb against C-terminus amino acids 1466-1480 coupled to keyhole limpet hemocyanin (MAb WC) stained dark cells predominantly. Staining by MAbs against the C-terminus was completely blocked by a C-terminus peptide. mRNA for CFTR was amplified by RT-PCR in both the duct and the secretory coil. In situ hybridization for CFTR mRNA after 3SR amplification indicates that mRNA is localized in the dark cells and perhaps also in the clear cell cytoplasm near the secretory coil. mRNA is present in both the luminal and basal duct cells. We conclude that CFTR is expressed equally well in both the duct and the secretory coil, suggesting that cAMP-dependent Cl(-) channels are involved in regulation of sweat secretion and duct absorption.     

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells

Chloride release from nonpigmented ciliary epithelial (NPE)cells is a final step in forming aqueous humor, and adenosine stimulates Cl transport by these cells. Whole cell patchclamping of cultured human NPE cells indicated that theA₃-selective agonist1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) stimulated currents (I_IB-MECA) by~90% at +80 mV. Partial replacement of external Clwith aspartate reduced outward currents and shifted the reversal potential (V_rev) from 23 ± 2 mV to0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusionwith the Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acidinhibited the currents. Partial Cl replacement withaspartate and NO₃, and perfusion with NPPB, hadsimilar effects on the swelling-activated whole cell currents(I_Swell). Partial cyclamate substitution for external Cl inhibited inward and outward currents of bothI_IB-MECA and I_Swell. Bothsets of currents also showed outward rectification and inactivation atlarge depolarizing potentials. The results are consistent with theconcept that A₃-subtype adenosine agonists and swellingactivate a common population of Cl channels.

     

Selectivity properties of a Na-dependent amino acid cotransport system in adult alveolar epithelial cells

We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by L-aspartic acid or L-proline. EC(50) values for L-lysine and L-histidine were 0.16 and 0.058 mM, respectively. The L-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. L-Serine, L-glutamine, and L-cysteine had EC(50) values of 0.095, 0.25, and 0.12 mM, respectively. L-Alanine had the highest affinity, with an EC(50) of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B(0,+). We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.     

Pyrimidinoceptors-mediated activation of Ca(2+)-dependent Cl(-) conductance in mouse endometrial epithelial cells

Previous studies have demonstrated the activation of endometrial Cl(-) secretion through P(2Y2) (P(2U)) purinoceptors by extracellular ATP. The present study further explored the presence of pyrimidine-sensitive receptors in the primary cultured mouse endometrial epithelial cells using the short-circuit current (I(SC)) and whole-cell patch-clamp techniques. UDP induced a transient increase in I(SC) in a concentration-dependent manner (EC(50) approximately 8.84 microM). The UDP-induced I(SC) was abolished after pretreating the epithelia with a calcium chelator, 1, 2-bis-(2-aminophenoxy)-ethane-N,N,N'N'tetraacetic acid-acetomethyl ester (BAPTA-AM), suggesting the dependence of the I(SC) on cytosolic free Ca(2+). The type of receptor involved was studied by cross-desensitization between ATP and UDP. ATP or UDP desensitized its subsequent I(SC) response. However, when ATP was added after UDP, or vice versa, a second I(SC) response was observed, indicating the activation of distinct receptors, possibly pyrimidine-sensitive receptors in addition to P(2Y2) (P(2U)) receptors. Similar results were observed in the patch-clamp experiments where UDP and ATP were shown to sequentially activate whole-cell current in the same cell. The UDP-activated whole-cell current exhibited outward rectification with delay activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. In addition, the UDP-evoked whole-cell current reversed near the equilibrium potential of Cl(-) in the presence of a Cl(-) gradient across the membrane, and was sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), indicating the activation of Ca(2+)-activated Cl(-) conductance. These characteristics were very similar to that of the ATP-activated whole-cell current. Taken together, our findings indicate the presence of distinct receptors, pyrimidinoceptors and P(2Y2) (P(2U)) receptors in mouse endometrial epithelial cells. These distinct receptors appear to converge on the same Ca(2+)-dependent Cl(-) channels.     

Regulation and function of the two-pore-domain (K2P) potassium channel Trek-1 in alveolar epithelial cells

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.     

The phorbol ester PMA and cyclic AMP activate different Cl(-) and HCO3(-) fluxes in C127 cells expressing CFTR

In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase C with the phorbol 12-myristate 13-acetate (PMA). Conversely, bicarbonate influx, determined by intracellular alkalinization of cells incubated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF-AM), was stimulated by cyclic AMP elevation, but not by PMA. Patch clamp analysis revealed that PMA activated a Cl(-) current with the typical biophysical characteristics of swelling-activated current and not of CFTR.     

cAMP regulation of Cl(-) and HCO(-)(3) secretion across rat fetal distal lung epithelial cells

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

Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis

CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.