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.

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.

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.

pH-regulated chloride secretion in fetal lung epithelia

2000 ◽  
Vol 278 (6) ◽  
pp. L1248-L1255 ◽  
Author(s):  
Carol J. Blaisdell ◽  
Rebecca D. Edmonds ◽  
Xi-Tao Wang ◽  
Sandra Guggino ◽  
Pamela L. Zeitlin
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Fetal Lung ◽  
Chloride Secretion ◽  
Lung Epithelial Cells ◽  
Apical Surface ◽  
Ussing Chambers ◽  
Lung Fluid ◽  
Lung Epithelial ◽  
Distal Lung

The fetal lung actively transports chloride across the airway epithelium. ClC-2, a pH-activated chloride channel, is highly expressed in the fetal lung and is located on the apical surface of the developing respiratory epithelium. Our goal was to determine whether acidic pH could stimulate chloride secretion in fetal rat distal lung epithelial cells mounted in Ussing chambers. A series of acidic solutions stimulated equivalent short-circuit current ( I eq) from a baseline of 28 ± 4.8 (pH 7.4) to 70 ± 5 (pH 6.2), 114 ± 12.8 (pH 5.0), and 164 ± 19.2 (pH 3.8) μA/cm2. These changes in I eq were inhibited by 1 mM cadmium chloride and did not result in large changes in [3H]mannitol paracellular flux. Immunofluorescent detection by confocal microscopy revealed that ClC-2 is expressed along the luminal surface of polarized fetal distal lung epithelial cells. These data suggest that the acidic environment of the fetal lung fluid could activate chloride channels contributing to fetal lung fluid production and that the changes in I eqseen in these Ussing studies may be due to stimulation of ClC-2.

Fetal lung cell-derived matrix alters distal lung epithelial ion transport

1995 ◽  
Vol 268 (5) ◽  
pp. L762-L771 ◽  
Author(s):  
O. M. Pitkanen ◽  
A. K. Tanswell ◽  
H. M. O'Brodovich
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Bathing Solution ◽  
Type I ◽  
Ussing Chambers ◽  
Lung Epithelial ◽  
Distal Lung

Extracellular matrix (ECM) synthesized by the fetal mesenchymal cells provides a supporting structure for the growing airways and is important for airway branching and in the differentiation of the primitive epithelium. We studied whether ECM, in addition to its structural role in lung interstitium, influences the ion transport of rat fetal distal lung epithelial cells (FDLE). FDLE monolayers were cultured on two different fetal mixed lung cell (MLC)-derived matrix preparations and studied in Ussing chambers. FDLE on MLC matrix had an increased resting equivalent short-circuit current (Ieq). Amiloride (10(-4) M apically) decreased the Ieq significantly in all the FDLE monolayers. The residual Ieq was significantly larger in FDLE grown on MLC matrix (increased by 150 and 80% under baseline and beta 2-agonist-stimulated conditions, respectively) than on control filters and filters coated with type I collagen, and type IV collagen, laminin, or fibronectin. The matrix produced by MLC isolated at an earlier gestational stage decreased the FDLE's sensitivity to amiloride. The increased amiloride-insensitive Ieq was only modestly affected by the Na+/K+/Cl- cotransport inhibitor bumetanide (10(-4) M basally) but was abolished when the [Cl-] of the bathing solution was reduced to 10 mM. These observations demonstrated that MLC elaborated ECM is able to change the nature of the ion transport of FDLE. ECM may be an important factor governing the ion transporting phenotype of fetal type II alveolar epithelial cells.

cAMP stimulates Na+ transport in rat fetal pneumocyte: involvement of a PTK- but not a PKA-dependent pathway

1999 ◽  
Vol 277 (4) ◽  
pp. L727-L736 ◽  
Author(s):  
Naomi Niisato ◽  
Yasushi Ito ◽  
Yoshinori Marunaka
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Short Circuit ◽  
Lung Epithelial Cells ◽  
Cell Shrinkage ◽  
Inhibitory Peptide ◽  
Stimulatory Action ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Dependent Pathway

To study a cAMP-mediated signaling pathway in the regulation of amiloride-sensitive Na+ transport in rat fetal distal lung epithelial cells, we measured an amiloride-sensitive short-circuit current (Na+ transport). Forskolin, which increases the cytosolic cAMP concentration, stimulated the Na+ transport. Forskolin also activated cAMP-dependent protein kinase (PKA). A β-adrenergic agonist and cAMP mimicked the forskolin action. PKA inhibitors KT-5720, H-8, and myristoylated PKA-inhibitory peptide amide-(14—22) did not influence the forskolin action. These results suggest that forskolin stimulates Na+ transport through a PKA-independent pathway. Furthermore, forskolin increased tyrosine phosphorylation of ∼70- to 80-, ∼97-, and ∼110- to 120-kDa proteins. Protein tyrosine kinase (PTK) inhibitors (tyrphostin A23 and genistein) abolished the forskolin action. Moreover, 5-nitro-2-(3-phenylpropylamino)benzoate (a Cl−-channel blocker) prevented the stimulatory action of forskolin on Na+ transport via abolishment of the forskolin-induced cell shrinkage and tyrosine phosphorylation. Based on these results, we conclude that forskolin (and cAMP) stimulates Na+ transport in a PTK-dependent but not a PKA-dependent pathway by causing cell shrinkage, which activates PTK in rat fetal distal lung epithelial cells.

Modulation of sodium transport in fetal alveolar epithelial cells by oxygen and corticosterone

2003 ◽  
Vol 284 (2) ◽  
pp. L376-L385 ◽  
Author(s):  
Ulrich H. Thome ◽  
Ian C. Davis ◽  
Susie Vo Nguyen ◽  
Brent Jay Shelton ◽  
Sadis Matalon
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Ussing Chambers ◽  
Alveolar Epithelial ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Postnatal Adaptation ◽  
Short Circuit Currents

Regulation of active Na+transport across fetal distal lung epithelial cells (FDLE) by corticosterone (CST), corticotropin-releasing hormone (CRH), and oxygen tension may be crucial for postnatal adaptation. FDLE isolated from 19-day rat fetuses (term: 22 days) were grown on permeable supports to confluent monolayers (duration 3 days) in 2.5, 5, 12, or 20% O2 with 5% CO2-balance N2 and mounted in Ussing chambers for measurement of short-circuit currents ( I sc). FDLE monolayers grown in 20% O2 had significantly higher levels of total I sc and of their amiloride-sensitive ( I amil) and ouabain-sensitive ( I ouab) components than hypoxic cells. Values (μA/cm2 ± SE) for 2.5–5% O2 and 20% O2 were, respectively, I sc5.3 ± 0.2 vs. 8.4 ± 0.3 ( P < 0.001), I amil 3.4 ± 0.2 vs. 4.3 ± 0.2 ( P < 0.01), and I ouab 3.4 ± 0.6 vs. 9.1 ± 0.6 ( P < 0.001). Addition of CST but not CRH to the culture medium at any O2concentration increased I amil. FDLE cells grown at 5% O2 expressed significantly lower levels of α-, β-, and γ-epithelial Na+ channel (ENaC), and of the α1-Na+-K+-ATPase, as determined by Western blotting. We conclude that higher O2concentrations increased total vectorial Na+ transport, and the function of Na+-K+-ATPase and apical amiloride-sensitive Na+ conductance, whereas CST only increased ENaC function.

Nitric oxide increases IL-8 gene transcription and mRNA stability to enhance IL-8 gene expression in lung epithelial cells

2004 ◽  
Vol 287 (4) ◽  
pp. L764-L773 ◽  
Author(s):  
Loretta Sparkman ◽  
Vijayakumar Boggaram
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Gene Transcription ◽  
Mrna Stability ◽  
Inflammatory Responses ◽  
Lung Epithelial Cells ◽  
Mrna Levels ◽  
Lung Epithelial

Interleukin (IL)-8, a C-X-C chemokine, is a potent chemoattractant and an activator for neutrophils, T cells, and other immune cells. The airway and respiratory epithelia play important roles in the initiation and modulation of inflammatory responses via production of cytokines and surfactant. The association between elevated levels of nitric oxide (NO) and IL-8 in acute lung injury associated with sepsis, acute respiratory distress syndrome, respiratory syncytial virus infection in infants, and other inflammatory diseases suggested that NO may play important roles in the control of IL-8 gene expression in the lung. We investigated the role of NO in the control of IL-8 gene expression in H441 lung epithelial cells. We found that a variety of NO donors significantly induced IL-8 mRNA levels, and the increase in IL-8 mRNA was associated with an increase in IL-8 protein. NO induction of IL-8 mRNA was due to increases in IL-8 gene transcription and mRNA stability. NO induction of IL-8 mRNA levels was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively, and 8-bromo-cGMP did not increase IL-8 mRNA levels. This indicated that NO induces IL-8 mRNA levels independently of changes in the intracellular cGMP levels. NO induction of IL-8 mRNA was significantly reduced by inhibitors of extracellular regulated kinase and protein kinase C. IL-8 induction by NO was also reduced by hydroxyl radical scavengers such as dimethyl sulfoxide and dimethylthiourea, indicating the involvement of hydroxyl radicals in the induction process. NO induction of IL-8 gene expression could be a significant contributing factor in the initiation and induction of inflammatory response in the respiratory epithelium.

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