O2-induced ENaC expression is associated with NF-κB activation and blocked by superoxide scavenger

1998 ◽  
Vol 275 (4) ◽  
pp. L764-L770 ◽  
Author(s):  
Bijan Rafii ◽  
A. Keith Tanswell ◽  
Gail Otulakowski ◽  
Olli Pitkänen ◽  
Rose Belcastro-Taylor ◽  
...  
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Nuclear Factor Κb ◽  
Lung Epithelial Cells ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Superoxide Scavenger ◽  
Transcription Factor Nuclear Factor

Cultured rat fetal distal lung epithelial cells (FDLEs), when switched from fetal (3%) to postnatal (21%) O2 concentrations, have increased epithelial Na+ channel (ENaC) mRNA levels and amiloride-sensitive Na+transport [O. Pitkänen, A. K. Tanswell, G. Downey, and H. O’Brodovich. Am. J. Physiol. 270 ( Lung Cell. Mol. Physiol. 14): L1060–L1066, 1996]. The mechanisms by which O2 mediates these effects are unknown. After isolation, FDLEs were kept at 3% O2 overnight, then switched to 21% O2 (3–21% O2 group) or maintained at 3% O2 (3–3% O2 group) for 48 h. The amiloride-sensitive short-circuit current ( I sc) in the 3–21% O2 group was double that in the 3–3% O2 group. Amiloride-sensitive I sc could not be induced by medium conditioned by 21% O2-exposed FDLEs but was reversed by returning the cells to 3% O2. Neither the cyclooxygenase inhibitor ibuprofen, liposome-encapsulated catalase, nor hydroperoxide scavengers (U-74389G or Trolox) blocked the O2-induced amiloride-sensitive I sc. In contrast, the cell-permeable superoxide scavenger tetramethylpiperidine- N-oxyl (TEMPO) eliminated the O2-induced increases in amiloride-sensitive I sc and ENaC mRNA levels. The switch from 3 to 21% O2 induced the transcription factor nuclear factor-κB, which could also be blocked by TEMPO. We conclude that 1) the O2-induced increase in amiloride-sensitive I sc is reversible and 2) the O2-induced increase in amiloride-sensitive I sc and ENaC mRNA levels is associated with activation of nuclear factor-κB and may be mediated, at least in part, by superoxide.

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide

1998 ◽  
Vol 274 (3) ◽  
pp. L378-L387 ◽  
Author(s):  
Jin Wen Ding ◽  
John Dickie ◽  
Hugh O’Brodovich ◽  
Yutaka Shintani ◽  
Bijan Rafii ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Cell Function ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Lung Epithelial Cells ◽  
Flufenamic Acid ◽  
Activated Macrophages ◽  
Lung Epithelial ◽  
Distal Lung

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( I sc) without affecting total I sc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the I sc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

Oxygen induction of epithelial Na+ transport requires heme proteins

2000 ◽  
Vol 278 (2) ◽  
pp. L399-L406 ◽  
Author(s):  
Bijan Rafii ◽  
Chris Coutinho ◽  
Gail Otulakowski ◽  
Hugh O'Brodovich
Keyword(s):  
Mrna Expression ◽  
Heme Proteins ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Iron Chelator ◽  
Mrna Levels ◽  
Hypoxic Conditions ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Iron Chelator Deferoxamine

Fetal distal lung epithelial (FDLE) cells exposed to a postnatal O2 concentration of 21% have higher epithelial Na+ channel (ENaC) mRNA levels and Na+ transport relative to FDLE cells grown in a fetal O2 concentration of 3%. To investigate the mechanism of this process, FDLE monolayers were initially cultured in 3% O2, and then some were switched to a 21% O2environment. Incubation of FDLE cells with the iron chelator deferoxamine, CoCl2, NiCl2, or an inhibitor of heme synthesis prevented or diminished the O2induction of amiloride-sensitive short-circuit current in FDLE cells. Similarly, defer- oxamine and cobalt prevented O2-induced ENaC mRNA expression. Exposure of FDLE cells grown under hypoxic conditions to carbon monoxide increased both ENaC mRNA expression and amiloride-sensitive short-circuit current. We therefore concluded that induction of ENaC mRNA expression and amiloride-sensitive Na+ transport in FDLE cells by a physiological increase in O2 concentration seen at birth requires iron and heme proteins.

Long-term terbutaline exposure stimulates α1-Na+-K+-ATPase expression at posttranscriptional level in rat fetal distal lung epithelial cells

2010 ◽  
Vol 298 (1) ◽  
pp. L96-L104 ◽  
Author(s):  
Muhammad S. Rahman ◽  
Shephali Gandhi ◽  
Gail Otulakowski ◽  
Wenming Duan ◽  
Aparna Sarangapani ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Lung Epithelial Cells ◽  
Intracellular Camp ◽  
Adult Type ◽  
Lung Epithelial ◽  
Distal Lung

Transepithelial Na+ transport through epithelial Na+ channels (ENaC) on the apical membrane and Na+-K+-ATPase activity on the basolateral membrane of distal lung epithelial cells are critical for alveolar fluid clearance. Acute exposure to β-adrenergic agonists stimulates lung fluid clearance by increasing Na+ transport. We investigated the effects of chronic exposure to the β2-adrenergic agonist terbutaline on the transepithelial Na+ transport in rat fetal distal lung epithelia (FDLE). FDLE monolayers exposed to 10−4 M terbutaline for 48 h had significantly increased propanolol-blockable transepithelial total and amiloride-sensitive short-circuit current ( Isc); however, when these chronically exposed monolayers were acutely exposed to additional β-agonists and intracellular cAMP upregulators, there was no further increase in Isc. Monolayers exposed to terbutaline for >48 h had Isc similar to control cells. Ouabain-sensitive Na+-K+-ATPase activity was increased in 48-h terbutaline-exposed FDLE whose apical membranes were permeabilized with nystatin. In contrast, terbutaline did not increase amiloride-sensitive apical membrane Isc in FDLE whose basolateral membranes were permeabilized with nystatin. Terbutaline treatment did not affect α-, β-, or γ-ENaC mRNA or α-ENaC protein steady-state levels, but increased total cellular levels and rate of synthesis of α1-Na+-K+-ATPase protein in FDLE in the absence of any change in α1-Na+-K+-ATPase mRNA. Total cellular β1-Na+-K+-ATPase mRNA and protein levels were not affected by terbutaline. These data suggest that FDLE have different responses from adult type II epithelial cells when chronically exposed to terbutaline, and their increased transepithelial Na+ transport occurs via a posttranscriptional increase in α1-Na+-K+-ATPase expression.

Inhibition by ethyl pyruvate of the nuclear translocation of nuclear factor-κB in cultured lung epithelial cells

2010 ◽  
Vol 23 (4) ◽  
pp. 308-315 ◽  
Author(s):  
Ayako Mizutani ◽  
Noriko Maeda ◽  
Seikichi Toku ◽  
Yoichiro Isohama ◽  
Kazuhiro Sugahara ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nuclear Translocation ◽  
Ethyl Pyruvate ◽  
Nuclear Factor Κb ◽  
Lung Epithelial Cells ◽  
Nuclear Factor ◽  
Lung Epithelial

Endotoxin-specific NF-κB activation in pulmonary epithelial cells harboring adenovirus E1A

1999 ◽  
Vol 277 (3) ◽  
pp. L523-L532 ◽  
Author(s):  
N. Keicho ◽  
Y. Higashimoto ◽  
G. P. Bondy ◽  
W. M. Elliott ◽  
J. C. Hogg ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Epithelial Cells ◽  
Tumor Necrosis ◽  
Inflammatory Mediator ◽  
Nuclear Factor Κb ◽  
Lung Epithelial Cells ◽  
Nuclear Factor ◽  
Adenovirus E1a ◽  
Lung Epithelial ◽  
Necrosis Factor

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-κB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-κB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c- jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-κB induction in these cells.

5-(N-Ethyl-N-isopropyl)amiloride sensitive Na+ currents in intact fetal distal lung epithelial cells

1993 ◽  
Vol 71 (1) ◽  
pp. 58-62 ◽  
Author(s):  
X. Wang ◽  
T. R. Kleyman ◽  
H. Tohda ◽  
Y. Marunaka ◽  
H. O'Brodovich
Keyword(s):  
Patch Clamp ◽  
Short Circuit ◽  
Primary Cultures ◽  
Alveolar Epithelium ◽  
Short Circuit Current ◽  
Lung Epithelial Cells ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Distal Lung

To determine whether primary cultures of rat fetal distal lung epithelium (FDLE) possessed L-type Na+ channels on their plasma membrane we performed experiments with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and other amiloride analogs. Short-circuit current (Isc) was decreased by the apical application of amiloride and benzamil, but was unaffected by 10 μM dimethylamiloride (DMA). EIPA decreased Isc when added to either the apical or basal sides. Greatest effects were seen with bilateral EIPA, where half-maximal effects occurred in the micromolar range. Measurements of intracellular pH with the fluorescent dye BCECF demonstrated that DMA impaired (IC50 = 71 nM) the ability of FDLE to recover from intracellular acidification. Nystatin perforated patch clamp techniques snowed that FDLE had nonrectifying Na+ currents but no detectable Cl− currents. The whole-cell currents were reversibly decreased by 20 μM concentrations of EIPA, benzamil, and amiloride but were unaffected by 20 μM DMA. These studies indicate that there are EIPA-sensitive Na+ conductances in intact FDLE and suggest the presence of L-type Na+ conductances on their apical membrane and EIPA-sensitive K+ channels on the basolateral membrane.Key words: Na+ channels, 5-(N-ethyl-N-isopropyl)amiloride, alveolar epithelium, whole-cell patch clamp, K+ channels.

cAMP regulation of Cl− and HCO 3 − secretion across rat fetal distal lung epithelial cells

2002 ◽  
Vol 282 (4) ◽  
pp. L650-L658 ◽  
Author(s):  
Ahmed Lazrak ◽  
Ulrich Thome ◽  
Carpantanto Myles ◽  
Janice Ware ◽  
Lan Chen ◽  
...  
Keyword(s):  
Short Circuit ◽  
Lung Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Anion Secretion ◽  
Ussing Chambers ◽  
Lung Epithelial ◽  
Cftr Protein ◽  
Distal Lung ◽  
Cystic Fibrosis Transmembrane

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 μA/cm2 and 1,018 ± 73 Ω · cm2, respectively (means ± SE; n = 12). Apical amiloride (10 μM) inhibited basal I sc by ∼50%. Subsequent addition of forskolin (10 μM) increased I sc from 3.9 ± 0.63 μA/cm2 to 7.51 ± 0.2 μA/cm2( n = 12). Basolateral bumetanide (100 μM) decreased forskolin-stimulated I sc from 7.51 ± 0.2 μA/cm2 to 5.62 ± 0.53, whereas basolateral 4,4′-dinitrostilbene-2,2′-disulfonate (5 mM), an inhibitor of HCO[Formula: see text] secretion, blocked the remaining I sc. Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl−- or HCO[Formula: see text]-free solutions; however, no response was seen using HCO[Formula: see text]- 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[Formula: see text] secretion across rat FDLE cells mediated via CFTR.

β-Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

2002 ◽  
Vol 282 (4) ◽  
pp. L621-L630 ◽  
Author(s):  
A. Collett ◽  
S. J. Ramminger ◽  
R. E. Olver ◽  
S. M. Wilson
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Anion Channel ◽  
Short Circuit Current ◽  
Lung Epithelial Cells ◽  
Disulfonic Acid ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Distal Lung ◽  
Stimulation Of

Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current ( I sc) that was inhibited (∼80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na+ from the apical solution. Isoprenaline increased the amiloride-sensitive I sc about twofold. Experiments in which apical membrane Na+ currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na+ conductance ( G Na+ ). Isoprenaline also increased apical Cl− conductance ( G Cl− ) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The isoprenaline-evoked changes in G Na+ and G Cl− could account for the changes in I sc observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I sc or G Na+ demonstrating that the rise in G Cl− is not essential to the stimulation of Na+ transport.

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