Update on the cellular and molecular aspects of cystic fibrosis transmembrane conductance regulator (CFTR) and male fertility.

CFTR protein regulates electrolyte and fluid transport in almost all tissues with exocrine function, including male reproductive tract. Mutation of CFTR gene causes cystic fibrosis (CF), which affects the function of several organs, and impairs male fertility. The role of CFTR protein in different compartments of male reproductive tract (testis, epididymis, sperm) as well as an impact of CFTR mutation(s) on male fertility phenotype is discussed in relation with the choice of optimal technique for Assisted Reproductive Techniques (ART) management.

Synthetic deoxynojirimycin derivatives bearing a thiolated, fluorinated or unsaturated N-alkyl chain: identification of potent α-glucosidase and trehalase inhibitors as well as F508del-CFTR correctors.

The synthesis of eleven 1-deoxynojirimycin (DNJ) derivatives presenting either a monofluoro, difluoro, thiolated or unsaturated N-alkyl chain of various length is described. Exploiting the unsaturated moiety on the nitrogen, fluorine has been introduced through a HF/SbF5 superacid catalysed hydrofluorination and thiol-ene click chemistry allowed introduction of sulfur. The synthetic derivatives have been tested for their ability to inhibit glycosidases and correct F508del-CFTR. Two of the unsaturated iminosugars exhibited potency similar to Miglustat as F508del-CFTR correctors. The thioalkyl iminosugars as well as the corresponding alkyl iminosugars demonstrated low micromolar α-glucosidases and trehalases inhibition. Introduction of fluorine abolished F508del-CFTR correction and trehalase inhibition.

N- and C-alkylation of seven-membered iminosugars generates potent glucocerebrosidase inhibitors and F508del-CFTR correctors.

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a ß-glucosidase and ß-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent ß-glucosidase, ß-galactosidase, α-L-rhamnosidase and ß-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 µM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.

[The impact of hypoxia on the ion channels in cystic fibrosis bronchial epithelial cells]. / Impact de l'hypoxie sur les canaux ioniques dans des cellules épithéliales bronchiques mucoviscidosiques.

Cystic fibrosis is a disease caused by a mutation on the CFTR gene coding for a chloride channel. The dominant mutation F508del eliminates the CFTR protein at the surface of epithelial cells, causing an accumulation of viscous mucus in the airways. In advanced stages of the disease, respiratory failure is associated with cellular hypoxia. Our project aims not only to describe the impact of hypoxia on ion channels and to highlight the underlying signaling pathways involved, but also to test the effectiveness of current CF treatments under the above-mentioned conditions.

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Identification of a novel water-soluble activator of wild-type and F508del CFTR: GPact-11a.

One of the major therapeutic strategy in cystic fibrosis aims at developing modulators of cystic fibrosis transmembrane conductance regulator (CFTR) channels. We recently discovered methylglyoxal alpha-aminoazaheterocycle adducts, as a new family of CFTR inhibitors. In a structure-activity relationship study, we have now identified GPact-11a, a compound able not to inhibit but to activate CFTR. Here, we present the effect of GPact-11a on CFTR activity using in vitro (iodide efflux, fluorescence imaging and patch-clamp recordings), ex vivo (short-circuit current measurements) and in vivo (salivary secretion) experiments. We report that GPact-11a: 1) is an activator of CFTR in several airway epithelial cell lines; 2) activates rescued F508del-CFTR in nasal, tracheal, bronchial, pancreatic cell lines and in human CF ciliated epithelial cells, freshly dissociated from lung samples; 3) stimulates ex vivo the colonic chloride secretion and increases in vivo the salivary secretion in cftr(+/+) but not cftr(-/-) mice; and 4) is selective for CFTR because its effect is inhibited by CFTR(inh)-172, GlyH-101, glibenclamide and GPinh-5a. To conclude, this work identifies a selective activator of wild-type and rescued F508del-CFTR. This nontoxic and water-soluble agent represents a good candidate, alone or in combination with a F508del-CFTR corrector, for the development of a CFTR modulator in cystic fibrosis.

9-phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels.

BACKGROUND AND PURPOSE: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. EXPERIMENTAL APPROACH: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. KEY RESULTS: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC(50) in the range of 2 x 10(-5) M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC(50). Channel inhibition was voltage independent. The IC(50) determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family. CONCLUSIONS AND IMPLICATIONS: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.

Characterization of cAMP dependent CFTR-chloride channels in human tracheal gland cells.

Human tracheal gland cells are believed to be a major site at the origin of cystic fibrosis. Since this disease is due to mutations in a protein called CFTR, we looked for the activity of CFTR in human tracheal gland cells in culture. We have identified CFTR-like chloride-selective channels as having a linear current voltage relationship and unitary conductance of 7 pS in these cells. In cell-attached patches, theophylline (1 mM), IBMX (1 mM), or a cocktail of dibutyryl cAMP (1 mM) and IBMX (0.1 mM) promoted the opening of channels. The unitary current had a reversal potential close to the cell resting potential. Replacement of choline by K+ or Na+ in the pipette solution was without effect on the current-voltage relationship, the reversal potential or the unitary conductance, which is consistent with the chloride selectivity of the channel. Channels were always found clustered and their opening probability was not noticeably dependent on membrane potential. This work therefore represents the first observation of a CFTR-like channel activity in submucosal gland cells.

Possible regulation of CFTR-chloride channels by membrane-bound phosphatases in pancreatic duct cells.

We have studied CFTR-Cl- channels in non-CF CAPAN-1 and in CFTR-transfected CFPAC-PLJ-CFTR-6 epithelial cells from human pancreas. Theophylline and IBMX induced the opening of cell-attached CFTR-Cl- channels. Theophylline, IBMX and the alkaline phosphatase (AP) inhibitor levamisole enhanced the activity of excised channels and reduced by 70-75% the apical membrane-associated APs activity. Okadaic acid had no effect on APs and channel activities. A polyclonal anti-alkaline phosphatase antibody (which detected apical APs) reduced APs activity and activated quiescent excised chloride channels. These results suggest that CFTR channels may be regulated by membrane-bound phosphatases.

Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel.

1. On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell-attached patch clamp and iodide efflux techniques. 2. Activation of CFTR channels was obtained with 3-mono, 1,3-di or 1,3,7-tri-substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8- or N9-position failed to open CFTR channels. 3. The CFTR chloride channel activity was blocked by glibenclamide (100 microM) but not by DIDS (100 microM). 4. Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20-1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. 5. We then synthesized a series of 3,7-dimethyl-1-alkyl xanthine derivatives; among them, 3,7-dimethyl-1-propyl xanthine and 3,7-dimethyl-1-isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7-dimethyl-1-(2-propenyl) xanthine and 3,7-dimethyl-1-(oxiranyl methyl) xanthine were inactive. 6. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

[CFTR and transepithelial ionic transport abnormalities in cystic fibrosis]. / CFTR et les anomalies des transports ioniques dans la mucoviscidose.

The genetic disease cystic fibrosis (CF) is caused by mutations of the CF gene and generates defective Cl- transport across the affected epithelium. Recent progress have been made to understand CFTR activity and regulation in epithelia and its role in the muco-ciliary clearance of airway. This revue-overviews the mechanisms of transepithelial ion transport, the role of CFTR in that process and the consequences for CF of CFTR mutations.

Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism.

BACKGROUND AND PURPOSE: The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. EXPERIMENTAL APPROACH: Human CF airway epithelial cells were treated with roscovitine 100 µM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. KEY RESULTS: Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. CONCLUSIONS AND IMPLICATIONS: Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.

From the vasodilator and hypotensive effects of an extract fraction from Agelanthus dodoneifolius (DC) Danser (Loranthaceae) to the active compound dodoneine.

AIM OF THE STUDY: Effects of the different fractions obtained by partition of ethanolic extract (EE) of Agelanthus dodoneifolius through column chromatography were investigated on rat blood pressure and aortic relaxation and compared to those observed in the presence of crude EE. MATERIALS AND METHODS: The acute hypotensive activity of EE, fractions and dodoneine, administrated intravenously, was evaluated in anaesthetized rats using the invasive method of blood pressure recording. Bioassay-guided fractionation using rat aorta pre-contracted by norepinephrine to monitor the relaxant activity led to the isolation of dodoneine. RESULTS: In normotensive rats, injection of EE (0.01-10 mg/kg) produced a dose-dependent decrease in both systolic and diastolic blood pressure without any significant change in heart rate. In a similar way, the EE (0.001-3 mg/mL) caused relaxation of rat pre-contracted aorta in a concentration-dependent manner. Fractionation of the EE afforded 14 fractions, F1-F14, that were tested on rat precontracted aortic rings. At the concentration level of 1 mg/mL, a maximum relaxation effect was observed for fractions F2-F5. F4 was the most effective to elicit a concentration-dependent relaxation effect with an ED(50)=160±1.1 µg/mL (n=5) and to decreased systolic and diastolic control pressure by 56.9% and 81.6% respectively. F4 contains most of the dihydropyranone dodoneine, with 93% of the sample mass. Dodoneine separated from this fraction was also able to decrease both systolic and diastolic arterial pressure by 32.5% and 38.7% at 100 µg/kg, respectively. CONCLUSION: For the first time, this study demonstrates the hypotensive property of the dodoneine present in Agelanthus dodoneifolius.

Expression and function of cystic fibrosis transmembrane conductance regulator in rat intrapulmonary arteries.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cyclic adenosine monophosphate (cAMP)-dependent chloride channel located mainly at the apical membrane of epithelial cells. In myocytes of pulmonary arteries, numerous chloride channels have been identified and described, but not the CFTR. Thus the presence and function of the CFTR was investigated in rat intrapulmonary arteries. CFTR expression, localisation and function were analysed in cultured smooth muscle cells using Reverse transcriptase (RT)-PCR and immunoprecipitation followed by protein kinase A phosphorylation, immunolocalisation and an iodide efflux assay, respectively. The role of the CFTR in pulmonary vasoreactivity was determined in arterial rings using an organ bath system. RT-PCR and immunoprecipitation analyses, as well as the immunolocalisation study, revealed the expression of CFTR gene transcripts and protein. The iodide efflux assay showed the existence of functional cAMP-, calcium- and volume-dependent chloride channels. Furthermore, the following effects were found: 1) inhibition of forskolin/genistein-activated iodide efflux by glibenclamide, diphenylamine-2-carboxylic acid and CFTR-specific inhibitor (CFTR(inh))-172; 2) activation of iodide efflux by the benzoquinolizinium derivative CFTR activators MPB-07 and MPB-91; and 3) inhibition of MPB-dependent efflux by CFTR(inh)-172. Finally, CFTR activators induced concentration-dependent vasorelaxation in rings preconstricted with phenylephrine, in the presence or absence of endothelium. The present results are the first to reveal functional cyclic adenosine monophosphate-regulated cystic fibrosis transmembrane conductance regulator contributing to endothelium-independent vasorelaxation in rat intrapulmonary arterial myocytes.

Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis.

BACKGROUND: Most patients with cystic fibrosis (CF) have a DeltaF508 mutation resulting in abnormal retention of mutant gene protein (DeltaF508-CFTR) within the cell. This study was undertaken to investigate DeltaF508-CFTR trafficking in native cells from patients with CF with the aim of discovering pharmacological agents that can move DeltaF508-CFTR to its correct location in the apical cell membrane. METHOD: Nasal epithelial cells were obtained by brushing from individuals with CF. CFTR location was determined using immunofluorescence and confocal imaging in untreated cells and cells treated with sildenafil. The effect of sildenafil treatment on CFTR chloride transport function was measured in CF15 cells using an iodide efflux assay. RESULTS: In most untreated CF cells DeltaF508-CFTR was mislocalised within the cell at a site close to the nucleus. Exposure of cells to sildenafil (2 hours at 37 degrees C) resulted in recruitment of DeltaF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased DeltaF508-CFTR trafficking in cells from individuals with CF with a single copy DeltaF508 (DeltaF508/4016ins) or with a newly described CF trafficking mutation (R1283M). CONCLUSIONS: The findings provide proof of principle for sildenafil as a DeltaF508-CFTR trafficking drug and give encouragement for future testing of sildenafil and related PDE5 inhibitors in patients with CF.

Phosphorylation-regulated low-conductance Cl- channels in a human pancreatic duct cell line.

A low-conductance Cl- channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mumol/l), dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mumol/l) and forskolin (10 mumol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23 degrees C and 12 pS at 37 degrees C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl- > I- >> > HCO3- > gluconate. In inside-out excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg2+). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl- channel was 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 100 mumol/l) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 100 mumol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mumol/l). These results demonstrate that the apical low-conductance Cl- channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative pharmacology of the activity of wild-type and G551D mutated CFTR chloride channel: effect of the benzimidazolone derivative NS004.

The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Using iodide ((125)I) efflux and whole-cell patch-clamp techniques we found dose-dependent stimulation of phosphorylated wt-CFTR channels by NS004 with an EC(50) approximately 11 microM. With non-phosphorylated CFTR, the effect of NS004 was apparent only at concentration >100 microM. In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 microM) nor NS004 (from 0.1 to 200 microM) added separately were able to stimulate channel activity. However, in the presence of 10 microM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC(50) approximately 1.5 microM. We also determined the half-maximal effective concentration of forskolin ( EC(50) approximately 3.2 microM) required to stimulate G551D channel activity in presence of 1.5 micro M NS004. No inhibitory effect was observed at high concentration of NS004 with both wt- and G551D-CFTR. Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time- and voltage-independent. The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 microM) and DPC (250 microM) but not by DIDS (200 microM) nor calixarene (100 nM). These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR.

Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.