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.

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.

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.

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

2000 ◽  
Vol 279 (1) ◽  
pp. G132-G138 ◽  
Author(s):  
Lane L. Clarke ◽  
Matthew C. Harline ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
John T. Turner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Receptor Activation ◽  
Biliary Disease ◽  
Anion Conductance ◽  
Intracellular Ca ◽  
Ph Stat ◽  
Chamber Studies

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO3 − secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y2 nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca2+-activated Cl− secretion. However, the value of this treatment in facilitating transepithelial HCO3 − secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca2+-dependent anion conductance during activation of luminal P2Y2receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ( I sc) that declined to a stable plateau phase lasting 30–60 min. The plateau I sc after UTP was Cl− independent, HCO3 − dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I sc and serosal-to-mucosal HCO3 − flux ( J s→m) during a 30-min period. Substitution of Cl− with gluconate in the luminal bath to inhibit Cl−/HCO3 −exchange did not prevent the increase in J s→mand I sc during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J s→m and I sc. We conclude that P2Y2 receptor activation results in a sustained (30–60 min) increase in electrogenic HCO3 − secretion that is mediated via an intracellular Ca2+-dependent anion conductance in CF gallbladder.

scholarly journals Assessment of Distinct Electrophysiological Parameters in Rectal Biopsies for the Choice of the Best Diagnosis/Prognosis Biomarkers for Cystic Fibrosis

2020 ◽  
Vol 11 ◽  
Author(s):  
Iris A. L. Silva ◽  
Aires Duarte ◽  
Fernando A. L. Marson ◽  
Raquel Centeio ◽  
Tereza Doušová ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Features ◽  
Ex Vivo ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Predictive Biomarker ◽  
Sweat Chloride ◽  
Best Parameter ◽  
Rectal Biopsies

Most cases of Cystic Fibrosis (CF) are diagnosed early in life. However, people with atypical CF forms pose diagnosis dilemmas, requiring laboratory support for diagnosis confirmation/exclusion. Ex vivo analysis of fresh rectal biopsies by Ussing chamber has been the best discriminant biomarker for CF diagnosis/prognosis so far. Here we aimed to evaluate different electrophysiological parameters from Ussing chamber analysis of rectal biopsies from people with CF (PwCF) to establish the one with highest correlations with clinical features as the best CF diagnosis/prognosis biomarker. We analyzed measurements of CFTR-mediated Cl– secretion in rectal biopsies from 143 individuals (∼592 biopsies), the largest cohort so far analyzed by this approach. New parameters were analyzed and compared with the previous biomarker, i.e., the IBMX (I)/Forskolin (F)/Carbachol (C)-stimulated short-circuit current (I’sc–I/F/C). Correlations with clinical features showed that the best parameter corresponded to voltage measurements of the I/F + (I/F/CCH) response (VI/F+I/F/C), with higher correlations vs. I’sc–I/F/C for: sweat chloride (59 vs. 52%), fecal elastase (69 vs. 55%) and lung function, measured by FEV1 (27 vs. 20%). Altogether data show that VI/F+I/F/C is the most sensitive, reproducible, and robust predictive biomarker for CF diagnosis/prognosis effectively discriminating classical, atypical CF and non-CF groups.

Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium

1991 ◽  
Vol 261 (2) ◽  
pp. C332-C341 ◽  
Author(s):  
N. J. Willumsen ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Open Circuit ◽  
Na Transport ◽  
Airway Epithelia ◽  
Human Nasal Epithelium ◽  
Epithelial Cultures

Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ transport in CF, CF nasal epithelial cultures were studied with double-barreled Na(+)-selective microelectrodes and the Ussing chamber technique. Intracellular Na+ activity (acNa) was 24.1 +/- 1.5 mM (n = 36), a value similar to acNa of normal nasal epithelial cells. Reduction of luminal [Na+] to 3 mM abolished Ieq and reduced acNa. Amiloride (10(-4) M) abolished Ieq but increased acNa from 20 +/- 2 to 36 +/- 7 mM (n = 10). Amiloride-induced increase in acNa was not affected by serosal [Na+] reduction but was blocked by preexposure to reduced luminal [Na+]. Amphotericin B increased Ieq during amiloride exposure, indicating that amiloride did not inhibit NA(+)-K(+)-ATPase. Ouabain abolished Ieq and slowly raised acNa. Reduction of serosal [Na+] led to a decrease in acNa that was blocked by bumetanide. It is concluded that 1) CF airway epithelia exhibit an increased apical membrane Na+ permeability, 2) acNa is regulated to a normal level in CF cells despite increased transcellular Na+ fluxes, 3) the abnormal increase in acNa in response to amiloride is dependent on luminal Na+, 4) Na+ is transported across the basolateral membrane by a bumetanide-sensitive cotransport mechanism, and 5) ouabain inhibits the basolateral Na(+)-K(+)-ATPase, causing slow dissipation of the chemical and electrical gradients across the cell membranes.

scholarly journals (NDRG2) Stimulates Amiloride-sensitive Na+ Currents in Xenopus laevis Oocytes and Fisher Rat Thyroid Cells

2007 ◽  
Vol 282 (38) ◽  
pp. 28264-28273 ◽  
Author(s):  
Mark O. Wielpütz ◽  
Il-Ha Lee ◽  
Anuwat Dinudom ◽  
Sheerazed Boulkroun ◽  
Nicolette Farman ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Feedback Inhibition ◽  
Short Circuit ◽  
Feedback Regulation ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Xenopus Laevis Oocytes ◽  
Thyroid Cells ◽  
Small Interference Rna ◽  
Rat Thyroid

Regulation of the epithelial sodium channel (ENaC) is highly complex and may involve several aldosterone-induced regulatory proteins. The N-Myc downstream-regulated gene 2 (NDRG2) has been identified as an early aldosterone-induced gene. Therefore, we hypothesized that NDRG2 may affect ENaC function. To test this hypothesis we measured the amiloride-sensitive (2 μm) whole cell current (ΔIami) in Xenopus laevis oocytes expressing ENaC alone or co-expressing ENaC and NDRG2. Co-expression of NDRG2 significantly increased ΔIami in some, but not, all batches of oocytes tested. An inhibitory effect of NDRG2 was never observed. Using a chemiluminescence assay we demonstrated that the NDRG2-induced increase in ENaC currents was accompanied by a similar increase in channel surface expression. The stimulatory effect of NDRG2 was preserved in oocytes maintained in a low sodium bath solution to prevent sodium feedback inhibition. These findings suggest that the stimulatory effect of NDRG2 is independent of sodium feedback regulation. Furthermore, the stimulatory effect of NDRG2 on ENaC was at least in part additive to that of Sgk1. A short isoform of NDRG2 also stimulated ΔIami. Overexpression of NDRG2 and ENaC in Fisher rat thyroid cells confirmed the stimulatory effect of NDRG2 on ENaC-mediated short-circuit current (ISC-ami). In addition, small interference RNA against NDRG2 largely reduced ISC-ami in Fisher rat thyroid cells. Our results indicate that NDRG2 is a likely candidate to contribute to aldosterone-mediated ENaC regulation.

Defective cholinergic Cl− secretion and detection of K+ secretion in rectal biopsies from cystic fibrosis patients

2000 ◽  
Vol 278 (4) ◽  
pp. G617-G624 ◽  
Author(s):  
M. Mall ◽  
A. Wissner ◽  
H. H. Seydewitz ◽  
J. Kuehr ◽  
M. Brandis ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Channel Blockers ◽  
K Secretion ◽  
Transepithelial Voltage ◽  
Human Airways ◽  
Rectal Biopsies

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl− secretion and an inverse response of the short-circuit current ( I sc) toward stimulation with carbachol (CCh). Alternative Cl− channels are found in airway epithelia and have been attributed to residual Cl− secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I sc upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca2+-dependent Cl− conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I sc were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I sc in CF rectal biopsies but caused a negative I sc in non-CF subjects. The CCh-induced negative I sc in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I sc was significantly enhanced in CF and was caused by activation of a luminal K+ conductance, as shown by the use of the K+ channel blockers Ba2+ and tetraethylammonium. Moreover, a cAMP-dependent luminal K+conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl− channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl−conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca2+- and cAMP-dependent K+ secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

scholarly journals Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

2006 ◽  
Vol 74 (5) ◽  
pp. 2937-2946 ◽  
Author(s):  
A. Ghosh ◽  
D. R. Saha ◽  
K. M. Hoque ◽  
M. Asakuna ◽  
S. Yamasaki ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Hela Cells ◽  
Histopathological Examination ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Toxin Gene ◽  
Dependent Manner ◽  
A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

scholarly journals Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 351 ◽  
Author(s):  
C. Tse ◽  
Julie In ◽  
Jianyi Yin ◽  
Mark Donowitz ◽  
Michele Doucet ◽  
...  
Keyword(s):  
Ion Transport ◽  
Serine Protease ◽  
Shiga Toxin ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Adult Stem Cell ◽  
Short Circuit Current ◽  
Watery Diarrhea ◽  
E Coli ◽  
Electrogenic Ion Transport

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

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