scholarly journals Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells

2016 ◽  
Vol 311 (2) ◽  
pp. L317-L327 ◽  
Author(s):  
Sharon M. Rymut ◽  
Claire M. Kampman ◽  
Deborah A. Corey ◽  
Tori Endres ◽  
Calvin U. Cotton ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Pediatric Population ◽  
Microtubule Dynamics ◽  
Cyclooxygenase Inhibitors ◽  
High Dose ◽  
Cell Periphery ◽  
Wild Type ◽  
Compound C

High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

scholarly journals Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells

2019 ◽  
Vol 316 (6) ◽  
pp. L1081-L1093
Author(s):  
Sharon M. Rymut ◽  
Binyu Lu ◽  
Aura Perez ◽  
Deborah A. Corey ◽  
Kata Lamb ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Adverse Events ◽  
Intracellular Transport ◽  
Adenosine Monophosphate ◽  
Cellular Level ◽  
Microtubule Dynamics ◽  
High Dose ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5′-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

scholarly journals The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel

2020 ◽  
Vol 21 (11) ◽  
pp. 4024 ◽  
Author(s):  
Ilona Gróf ◽  
Alexandra Bocsik ◽  
András Harazin ◽  
Ana Raquel Santa-Maria ◽  
Gaszton Vizsnyiczai ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Sodium Bicarbonate ◽  
Vascular Endothelial Cells ◽  
Bronchial Epithelium ◽  
Barrier Properties ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Bronchial Epithelial

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

scholarly journals Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

2011 ◽  
Vol 301 (4) ◽  
pp. L557-L567 ◽  
Author(s):  
Ahmed Lazrak ◽  
Asta Jurkuvenaite ◽  
Lan Chen ◽  
Kim M. Keeling ◽  
James F. Collawn ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Alveolar Epithelial ◽  
Cystic Fibrosis Transmembrane

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous ( Cftr +/−), knockout ( Cftr −/−), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (Po): wild-type=0.21 ± 0.015: Cftr +/−=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr −/−=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the Po and number of channels in ATII cells of wild-type, Cftr +/−, and ΔF508, but not in Cftr −/− mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr +/− and Cftr −/− mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.

scholarly journals Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

2012 ◽  
Vol 303 (2) ◽  
pp. L97-L106 ◽  
Author(s):  
Shilpa Nimishakavi ◽  
Marina Besprozvannaya ◽  
Wilfred W. Raymond ◽  
Charles S. Craik ◽  
Dieter C. Gruenert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Selective Inhibition ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+ channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

Whole cell Cl- conductances in mouse choroid plexus epithelial cells do not require CFTR expression

1997 ◽  
Vol 272 (6) ◽  
pp. C1899-C1907 ◽  
Author(s):  
J. D. Kibble ◽  
C. Garner ◽  
W. H. Colledge ◽  
S. Brown ◽  
H. Kajita ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Choroid Plexus ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Significant Difference ◽  
Choroid Plexus Epithelial

Whole cell patch-clamp studies were performed with tissue isolated from the cystic fibrosis (CF) transgenic Cftrm1cam mouse, to determine whether anion currents in choroid plexus epithelial cells require the expression of cystic fibrosis transmembrane conductance regulator (CFTR). Inclusion of 0.25 mM adenosine 3',5'-cyclic monophosphate (cAMP) and 375 nM protein kinase A (PKA) in the pipette solution caused a significant activation of a Cl(-)-selective, inward-rectifying conductance in cells from wild-type and CF mice. The small, outward currents observed in wild-type and CF animals, however, were not activated by cAMP-PKA. There were no significant differences in the size of currents between wild-type, heterozygote, and CF cells in the presence or absence of cAMP-PKA. A second whole cell conductance was activated when cells from wild-type mice were swollen. These volume-activated currents were Cl- selective and exhibited outward rectification. They were Ca2+ independent and ATP dependent and blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The volume-activated channels were also activated in CF mutant cells, and there was no significant difference in the size of the volume-activated currents between wild-type, heterozygote, and CF cells. It is concluded that CFTR neither contributes to the whole cell conductance nor regulates the other anion conductances in choroid plexus epithelial cells.

Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents

1994 ◽  
Vol 266 (5) ◽  
pp. C1464-C1477 ◽  
Author(s):  
E. M. Schwiebert ◽  
T. Flotte ◽  
G. R. Cutting ◽  
W. B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Wild Type ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Cl Channel ◽  
Cl Channels

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

scholarly journals Augmentation of CFTR maturation by S-nitrosoglutathione reductase

2016 ◽  
Vol 310 (3) ◽  
pp. L263-L270 ◽  
Author(s):  
Khalequz Zaman ◽  
Victoria Sawczak ◽  
Atiya Zaidi ◽  
Maya Butler ◽  
Deric Bennett ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Reductase Activity ◽  
Subcellular Location ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial ◽  
Human Airway ◽  
Human Airway Epithelial Cells

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

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