Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium

Sheppard, David N.; Carson, Mark R.; Ostedgaard, Lynda S.; Denning, Gerene M.; Welsh, Michael J.

doi:10.1152/ajplung.1994.266.4.l405

Cited by 66 publications

(79 citation statements)

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“…If no transporter is present, iodide cannot enter the cell because a balancing flow of chloride is not possible. FRT cells are relatively impermeable to anions because they lack key epithelial ion channels (including cystic fibrosis transmembrane conductance regulator (CFTR), the channel defective in CF 38 ). However, in the presence of an anionophore, iodide influx occurs leading to YFP fluorescence quenching ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…FRT is used especially for CFTR research because it forms an epithelium which lacks endogenous CFTR expression. 38 HeLa cells are widely used for cell biology studies, and were employed for toxicity testing on calixpyrrole 3 25 showed quite strong inhibition for HeLa cells, but the effect of 13 was only minor. As 13 is a stronger anionophore than 12, it seems likely that the toxic effects of the latter are not purely due to anion transport.…”

Section: (Figure 3)mentioning

confidence: 99%

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Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

Valkenier

Judd³

et al. 2015

Nature Chem

151

160

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The transport of anions across cell membranes has become an important goal for supramolecular chemistry [1][2][3][4] . It is well-known that cation carriers (cationophores) can serve as antibiotics and toxins 5,6 , and it seems likely that anion carriers might also show useful biological activity. However, suitable anionophores have only recently become available 7-10 , and the nature of their biological effects is still in question. A particular hope is that anionophores might be used to replace the activity of endogenous anion channels which are missing or defective 11,2 . Such deficiencies underlie a number of medical conditions including Best disease, Startle disease, Bartter's syndrome and, most notably, the widespread lifeshortening genetic disease cystic fibrosis (CF) 12,13 .If anionophores are to be used to treat these "channelopathies", it must be shown that they can be delivered to cells in sufficient quantities to produce substantial effects, of the same order of magnitude as endogenous anion channels, and that these quantities are not toxic to the cells. However, while a wide variety of anionophores have been studied in synthetic membranes (principally large unilamellar vesicles, or LUVs), the range of systems subjected to biological investigations is still quite limited. Moreover, from the point of view of CF treatment, the results thus far have been mixed. On the positive side, a few systems have been tested in whole cells, epithelia or (in one case) genetically modified mice 14 , using electrophysiological methods such as patch-clamp and Ussing chamber, and shown to induce anion conduction without obvious toxic effects. These anionophores include synthetic 3 peptides conceptually related to natural anion channels 11,[15][16][17] , as well as the steroid-based system 1 from the authors' laboratory 18 , and other small organic molecules 14,19,20 .Less encouragingly, a number of molecules showing anion transport in LUVs have tested positive for anti-cancer cytotoxicity (potentially valuable, but incompatible with CF treatment) 8,[21][22][23][24][25] . Many of these systems, including the well-studied prodigiosin 2 26 , transport protons as well as anions 8,[21][22][23] . Intracellular acidification can lead to apoptosis 26 , so in these cases proton transport could underlie toxicity. On the other hand, a recent study on calixpyrroles 3 suggested that anion transport as such could be cytotoxic 25 . As the transport activity of 3 was only weak, this raises the concern that powerful anionophores might be highly toxic and thus unsuitable for CF treatment. Thus far the only biological testing of these "1,5-diaxial" systems has been the early study on 1, referred to above 18 . The assay involved the application of 1 to the apical membrane of oriented Madin Darby canine kidney cell (MDCK) epithelia, mounted in an Ussing chamber, and measurement of the resulting electrical current caused by Cl − transport. The methodology is well-established, but not so convenient for screening large numbers of compounds.Mor...

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Section: Resultsmentioning

confidence: 99%

Section: (Figure 3)mentioning

confidence: 99%

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

Valkenier

Judd³

et al. 2015

Nature Chem

151

160

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“…This proposal is supported by the observation that inhibitors of cloned pCFTR also inhibit electrogenic anion secretion by the possum ileum. When expressed in the FRT cell line, a model epithelium (Sheppard et al, 1994), cloned pCFTR has a distinct inhibitor profile. The non-specific anion channel blocker, NPPB (Cabantchik and Greger, 1992), and the specific CFTR inhibitor, GlyH101 (Muanprasat et al, 2007), potently inhibit pCFTR.…”

Section: Discussionmentioning

confidence: 99%

“…After permeabilisation with amphotericin B, the basolateral membrane was electrically eliminated so that changes in currents induced by drugs reflect the changes in conductance of the apical membrane, where pCFTR is primarily located. Therefore, the measured transepithelial current is due to Cl -flow through pCFTR in the apical membrane and this Cl -current (I Cl ) is an index of pCFTR activity (Sheppard et al, 1994). pCFTR activity was stimulated with the addition of mucosal and serosal forskolin (10moll -1 ) and 3-isobutyl-1-methylxanthine (IBMX; 100moll -1 ).…”

Section: Measurements Of Epithelial Transportmentioning

confidence: 99%

“…To characterise the pharmacological profile of cloned pCFTR, confluent monolayers of FRT cells stably expressing pCFTR were mounted in Ussing chambers and pCFTR activity was quantified as the cAMP-activated I Cl measured across these monolayers following exposure to a Cl -gradient and permeabilisation of the basolateral membrane with amphotericin (Sheppard et al, 1994). Under these conditions, the addition of forskolin (10moll -1 ) plus IBMX (100moll -1 ) resulted in the stimulation of I Cl in confluent monolayers of cells transfected with pCFTR, whereas there was no increase in I Cl in monolayers grown from cells transfected with the empty vector (Fig.5A,B).…”

Section: Pharmacology Of Pcftr and Ileal Hco 3 -Secretionmentioning

confidence: 99%

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The distribution and expression of CFTR restricts electrogenic anion secretion to the ileum of the brushtail possum, Trichosurus vulpecula

Gill

Bartolo

Demmers

et al. 2011

Journal of Experimental Biology

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SUMMARYIn eutherian mammals, fluid secretion is essential for intestinal function. This is driven by electrogenic Cl -secretion, which involves a NaK2Cl cotransporter (NKCC1) in the enterocyte basolateral membrane and the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, in the possum ileum, NKCC1 expression is low and secretagogues stimulate electrogenic HCO 3 -secretion driven by a basolateral NaHCO 3 cotransporter (pNBCe1). Here we investigated whether electrogenic anion secretion occurs in possum duodenum and jejunum and determined the role of CFTR in possum intestinal anion secretion. Prostaglandin E 2 (PGE 2 ) and forskolin stimulated a large increase in ileal short-circuit current (I sc ), consistent with electrogenic HCO 3 -secretion, but had little effect on the duodenal and jejunal I sc . Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH101) inhibited cloned possum CFTR in cultured cells and the PGE 2 -stimulated ileal I sc , implicating CFTR in ileal HCO 3 -secretion. Consistent with this, CFTR is expressed in the apical membrane of ileal crypt and lower villous cells, which also express pNBCe1 in the basolateral membrane. In contrast, duodenal and jejunal CFTR expression is low relative to the ileum. Jejunal pNBCe1 expression is also low, whereas duodenal and ileal pNBCe1 expression are comparable. All regions have low NKCC1 expression. These results indicate that cAMP-dependent electrogenic Cl -secretion does not occur in the possum small intestine because of the absence of CFTR and NKCC1. Furthermore, CFTR functions as the apical anion conductance associated with HCO 3 -secretion and its distribution limits electrogenic HCO 3 -secretion to the ileum.

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CFTR interacts with Hsp90 and regulates the phosphorylation of AKT and ERK1/2 in colorectal cancer cells

et al. 2019

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Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. CF cells and tissues exhibit various mitochondrial abnormalities. However, the underlying molecular mechanisms remain elusive. Here, we examined the mechanisms through which CFTR regulates Bcl‐2 family proteins, which in turn regulate permeabilization of the mitochondrial outer membrane. Notably, inhibition of CFTR activated Bax and Bad, but inhibited Bcl‐2. Moreover, degradation of phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) and AKT increased significantly in CFTR‐knockdown cells. Dysfunction of CFTR decreased heat‐shock protein 90 ( Hsp90 ) mRNA levels, and CFTR was found to interact with Hsp90. Inhibition of Hsp90 by SNX‐2112 induced the degradation of phosphorylated AKT and ERK1/2 in Caco2 and HRT18 cells. These findings may help provide insights into the physiological role of CFTR in CF‐related diseases.

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Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium

Cited by 66 publications

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Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

The distribution and expression of CFTR restricts electrogenic anion secretion to the ileum of the brushtail possum, Trichosurus vulpecula

CFTR interacts with Hsp90 and regulates the phosphorylation of AKT and ERK1/2 in colorectal cancer cells

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