CFTR activation in human bronchial epithelial cells by novel benzoflavone and benzimidazolone compounds

2003 ◽  
Vol 285 (1) ◽  
pp. L180-L188 ◽  
Author(s):  
Emanuela Caci ◽  
Chiara Folli ◽  
Olga Zegarra-Moran ◽  
Tonghui Ma ◽  
Mark F. Springsteel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
High Throughput Screening ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Thyroid Cells ◽  
Bronchial Epithelial ◽  
Rat Thyroid ◽  
Better Than

Activators of the CFTR Cl- channel may be useful for therapy of cystic fibrosis. Short-circuit current ( Isc) measurements were done on human bronchial epithelial cells to characterize the best flavone and benzimidazolone CFTR activators identified by lead-based combinatorial synthesis and high-throughput screening. The 7,8-benzoflavone UCcf-029 was a potent activator of Cl- transport, with activating potency (<1 μM) being much better than other flavones, such as apigenin. The benzimidazolone UCcf-853 gave similar Isc but with lower potency (5–20 μM). In combination, the effect induced by maximal UCcf-029 and UCcf-029, UCcf-853, and apigenin increased strongly with increasing basal CFTR activity: for example, Kd for activation by UCcf-029 decreased from >5 to <0.4 μM with increasing basal Isc from ∼4 μA/cm2 to ∼12 μA/cm2. This dependence was confirmed in permeabilized Fischer rat thyroid cells stably expressing CFTR. Our results demonstrate efficacy of novel CFTR activators in bronchial epithelia and provide evidence that activating potency depends on basal CFTR activity.

An electrogenic amino acid transporter in the apical membrane of cultured human bronchial epithelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. L917-L923 ◽  
Author(s):  
Luis J. V. Galietta ◽  
Luciana Musante ◽  
Leila Romio ◽  
Ubaldo Caruso ◽  
Annarita Fantasia ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Bronchial Epithelial Cells ◽  
Maximal Effect ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial

We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolaterall-arginine (10–1,000 μM) increased the short-circuit current. Maximal effect and EC50were ∼3.5 μA/cm2and 80 μM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor ( NG-nitro-l-arginine methyl ester) did not decrease the arginine-dependent current. Apicall-lysine,l-alanine, andl-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50values ranging from 26 to 971 μM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.

Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells

2002 ◽  
Vol 282 (2) ◽  
pp. L226-L236 ◽  
Author(s):  
Henry Danahay ◽  
Hazel Atherton ◽  
Gareth Jones ◽  
Robert J. Bridges ◽  
Christopher T. Poll
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Anion Conductance

Interleukin (IL)-13 has been associated with asthma, allergic rhinitis, and chronic sinusitis, all conditions where an imbalance in epithelial fluid secretion and absorption could impact upon the disease. We have investigated the effects of IL-13 on the ion transport characteristics of human bronchial epithelial cells cultured at an apical-air interface. Ussing chamber studies indicated that 48 h pretreatment with IL-13 or IL-4 significantly reduced the basal short-circuit current ( I sc) and inhibited the amiloride-sensitive current by >98%. Furthermore, the I scresponses were increased by more than six- and twofold over control values when stimulated with UTP or forskolin, respectively, after cytokine treatment. The IL-13-enhanced response to UTP/ionomycin was sensitive to bumetanide and DIDS and was reduced in a low-chloride, bicarbonate-free solution. Membrane permeablization studies indicated that IL-13 induced the functional expression of an apical Ca2+-activated anion conductance and that changes in apical or basolateral K+ conductances could not account for the increased I sc responses to UTP or ionomycin. The results indicate that IL-13 converts the human bronchial epithelium from an absorptive to a secretory phenotype that is the result of loss of amiloride-sensitive current and an increase in a DIDS-sensitive apical anion conductance.

Protease-activated receptor-2-mediated inhibition of ion transport in human bronchial epithelial cells

2001 ◽  
Vol 280 (6) ◽  
pp. C1455-C1464 ◽  
Author(s):  
Henry Danahay ◽  
Louise Withey ◽  
Christopher T. Poll ◽  
Stan F. J. van de Graaf ◽  
Robert J. Bridges
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Bronchial Epithelial Cells ◽  
Transient Increase ◽  
Liquid Volume ◽  
Apical Surface ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Protease Activated Receptor 2

A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current ( I sc), followed by a sustained inhibition of amiloride-sensitive I sc. These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in I sc was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.

scholarly journals Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

2011 ◽  
Vol 301 (4) ◽  
pp. L587-L597 ◽  
Author(s):  
Louise C. Pyle ◽  
Annette Ehrhardt ◽  
Lisa High Mitchell ◽  
LiJuan Fan ◽  
Aixia Ren ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
High Throughput Screening ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Regulatory Domain ◽  
Thyroid Cells ◽  
Binding Domains ◽  
Mechanistic Basis ◽  
Rat Thyroid

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UCCF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UCCF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o− and Fischer rat thyroid cells, expressing various CFTR forms. UCCF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UCCF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

scholarly journals Culture with apically applied healthy or disease sputum alters the airway surface liquid proteome and ion transport across human bronchial epithelial cells.

2021 ◽  
Author(s):  
Maximillian Woodall ◽  
Boris Reidel ◽  
Mehmet Kesimer ◽  
Robert Tarran ◽  
Deborah L Baines
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Bronchial Epithelial Cells ◽  
Airway Surface Liquid ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Surface Liquid

Airway secretions contain many signalling molecules and peptides/proteins that are not found in airway surface liquid (ASL) generated by normal human bronchial epithelial cells (NHBE) in vitro. These play a key role in innate defence and mediate communication between the epithelium, immune cells and the external environment. We investigated how culture of NHBE with apically applied secretions from healthy or disease (Cystic Fibrosis, CF) lungs affected epithelial function with a view to providing better in vitro models of the in vivo environment. NHBE from 6-8 different donors were cultured at air-liquid interface (ALI), with apically applied sputum from normal healthy donors (NLS) or CF donors (CFS) for 2-4 hours, 48 hours or with sputum reapplied over 48 hours. Proteomic analysis was carried out on the sputa and on NHBE ASL before and after culture with sputa. Transepithelial electrical resistance (TEER), short circuit current (Isc) and changes to ASL height were measured. There were 71 proteins common to both sputa but not ASL. The protease:protease inhibitor balance was increased in CFS compared to NLS and ASL. Culture of NHBE with sputa for 48 hours identified additional factors not present in NLS, CFS or ASL alone. Culture with either NLS or CFS for 48 hours increased CFTR activity, calcium activated chloride channel (CaCC) activity and changed ASL height. These data indicate that culture with healthy or disease sputum changes the proteomic profile of ASL and ion transport properties of NHBE and this may increase physiological relevance when using in vitro airway models.

Phospholipase C controls chloride-dependent short-circuit current in human bronchial epithelial cells

2020 ◽  
Author(s):  
Chloé Grebert ◽  
Frederic Becq ◽  
Clarisse Vandebrouck
Keyword(s):  
Epithelial Cells ◽  
Phospholipase C ◽  
Chloride Channels ◽  
Short Circuit ◽  
Ion Homeostasis ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bronchial Epithelial Cells ◽  
Airway Epithelial ◽  
Bronchial Epithelial

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over expressing WT‑ or F508del‑CFTR. Western blot analysis shows expression of the three endogenous PLC isoforms PLCd1, PLCg1 and PLCb3 in 16HBE14o‑ cells. In 16HBE14o‑ cells we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCb3 and PLCg1 in CFTR-dependent short-circuit current activated by forskolin, but not PLCd1. In CFBE-WT CFTR and corrected CFBE‑F508del CFTR cells, PLCb3 silencing also inhibits CFTR‑dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR‑dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells.

Partial correction of defective Cl− secretion in cystic fibrosis epithelial cells by an analog of squalamine

2001 ◽  
Vol 281 (5) ◽  
pp. L1164-L1172 ◽  
Author(s):  
Canwen Jiang ◽  
Edward R. Lee ◽  
Mathieu B. Lane ◽  
Yong-Fu Xiao ◽  
David J. Harris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Synthetic Analog ◽  
Airway Epithelial ◽  
Thyroid Cells ◽  
Rat Thyroid ◽  
Partial Correction

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl− transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl− transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl− gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ∼30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl− currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl− solution, indicating restoration of Cl− secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl− secretion.

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