3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone and Cystic-Fibrosis

Cystic fibrosis-related diabetes (CFRD) worsens CF lung disease leading to early mortality. Loss of beta cell area, even without overt diabetes or pancreatitis is consistently observed. We investigated whether short-term CFTR inhibition was sufficient to impact islet morphology and function in otherwise healthy mice. CFTR was inhibited in C57BL/6 mice via 8-day intraperitoneal injection of CFTRinh172. Animals had a 7-day washout period before measures of hormone concentration or islet function were performed. Short-term CFTR inhibition increased blood glucose concentrations over the course of the study. However, glucose tolerance remained normal without insulin resistance. CFTR inhibition caused marked reductions in islet size and in beta cell and non-beta cell area within the islet, which resulted from loss of islet cell size rather than islet cell number. Significant reductions in plasma insulin concentrations and pancreatic insulin content were also observed in CFTR-inhibited animals. Temporary CFTR inhibition had little long-term impact on glucose-stimulated, or GLP-1 potentiated insulin secretion. CFTR inhibition has a rapid impact on islet area and insulin concentrations. However, islet cell number is maintained and insulin secretion is unaffected suggesting that early administration of therapies aimed at sustaining beta cell mass may be useful in slowing the onset of CFRD.

Topics: Animals; Benzoates; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diabetes Mellitus; Disease Models, Animal; Humans; Insulin; Insulin-Secreting Cells; Male; Mice; Thiazolidines

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to almost total absence of CFTR at the plasma membrane, a defect potentially corrected via drug-based therapies. Herein, we report the first proof-of-principle study of a noninvasive imaging probe able to detect CFTR at the plasma membrane. We radiolabeled the CFTR inhibitor, CFTR

Topics: Benzoates; Cell Line; Cell Membrane; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diamines; Humans; Mutation; Pyrazoles; Radiopharmaceuticals; Technetium; Thiazolidines

To date, three β-adrenoceptor (β-AR) subtypes have been identified, but only β(1)-ARs and β(2)-ARs have been characterized in human lungs. Moreover, β(2)-ARs physically interact with the cystic fibrosis transmembrane conductance regulator (CFTR) through the Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) protein. β(3)-ARs, which stimulate CFTR activity in transfected cells, have not been identified in human lungs. This study aimed (1) to characterize the presence of β-AR subtypes, especially β(3)-AR, in human bronchi, and (2) to compare their expression as well as that of NHERF1 in non-cystic fibrosis (CF) versus advanced CF lung samples. In human non-CF bronchi, β(1)-AR, β(2)-AR, β(3)-AR, and NHERF1 transcripts and proteins were expressed mainly in bronchial epithelial cells. Those results were strengthened by the native expression of β(1)-AR, β(2)-AR, and β(3)-AR in a human epithelial cell line, 16HBE14o(-). All β-AR subtypes stimulated CFTR activity. In CF bronchi, we demonstrated β(1)-AR and β(3)-AR overexpression, and NHERF1 and β(2)-AR underexpression. The origin of this protein remodeling (involving the physical or functional absence of CFTR, infection, inflammation, or high adrenergic tone) deserves further investigation. These results evidence for the first time, to the best of our knowledge, the presence of β(3)-ARs in human bronchi, and suggest their usefulness as a putative new pharmacologic target in lung diseases where fluid homeostasis is altered. Furthermore, NHERF1 may be a new therapeutic target in patients with CF, to facilitate the trafficking of mutated CFTR to plasma membrane.

Topics: Adrenergic beta-Agonists; Adult; Aged; Albuterol; Benzoates; Bronchi; Case-Control Studies; Cell Line; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dobutamine; Female; Gene Expression Regulation; Humans; Male; Middle Aged; Phosphoproteins; Prazosin; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Receptors, Adrenergic, beta-3; RNA, Messenger; Sodium-Hydrogen Exchangers; Thiazolidines; Young Adult

1. Cystic fibrosis transmembrane conductance regulator (CFTR) is well known for its role in the cystic fibrosis (CF). Recent studies have shown that CF patients and CFTR-deficient mice exhibit a severe abnormal skeletal phenotype, indicating that CFTR may play a role in bone development and pathophysiological processes. However, it is not known whether CFTR has a direct or indirect effect on bone formation. The aim of the present study was to detect the expression and function of CFTR in mouse chondrocytes. 2. Reverse transcription-polymerase chain reaction, western blotting and immunofluorescence were used to characterize the expression of CFTR in primary isolated mouse chondrocytes. Expression of CFTR mRNA and protein was detectable in mouse chondrocytes. Importantly, whole-cell patch-clamp analysis demonstrated that CFTR in mouse chondrocytes is functional as a cAMP-dependent Cl(-) channel that is inhibited by CFTRinh-172. 3. Thus, the results of the present study demonstrate that functional CFTR is expressed in mouse chondrocytes, which offers essential evidence for the potential direct role of CFTR in physiological and pathological processes of bone.

Topics: Animals; Animals, Newborn; Benzoates; Cells, Cultured; Chondrocytes; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Male; Membrane Transport Modulators; Mice; Osteogenesis; Patch-Clamp Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidines; Transfection

Cystic fibrosis (CF) is a chronic inflammatory lung disease characterized by polymorphonuclear neutrophil (PMN)-dominated airway inflammation. Defective apoptosis might explain PMN persistence at these inflammation sites. We previously reported that in CF patients PMN underwent delayed apoptosis, which was not always related to their infectious state and independent of the type of CF transmembrane regulator (CFTR) mutation. To understand the role of infection and PMN apoptosis in CF, PMN apoptosis was investigated in CF parents who are obligate heterozygotes for the CFTR mutation but without chronic bacterial infection. They also demonstrated delayed PMN apoptosis compared with healthy controls, as assessed by annexin-V labeling and caspase-3 cleavage. Diamide, a direct thiol-oxidizing agent, potentiated PMN apoptosis in controls and CF patients, resulting in similar levels of constitutive and Fas-potentiated apoptosis. The cyclin-dependent kinase inhibitor roscovitine provided another approach to restore normal PMN apoptosis. However, the selective CFTR inhibitor CFTR(Inh172) did not affect PMN apoptosis in control subjects. Apparently, the dysregulation of CF PMN is not only a consequence of the chronic infectious state in CF children but might also be related to CF 'intrinsic' factors. Restoration of normal PMN apoptosis by cellular redox modulation or roscovitine opens new research avenues to decrease PMN-mediated inflammation in CF.

Topics: Adolescent; Apoptosis; Bacterial Infections; Benzoates; Caspase 3; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diamide; Female; Heterozygote; Homozygote; Humans; Male; Neutrophils; Oxidation-Reduction; Purines; Roscovitine; Thiazolidines

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.. Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.. Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.. The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.

Topics: Animals; Benzoates; Binding Sites; Cell Line; Cell Membrane; Cholesterol; Cholesterol Oxidase; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Electrochemical Techniques; Epithelial Cells; Genotype; Humans; Hydroxymethylglutaryl-CoA Synthase; Kinetics; Mass Spectrometry; Mice; Mice, Inbred CFTR; Microelectrodes; Mutation; Nasal Mucosa; Phenotype; Promoter Regions, Genetic; Respiratory Mucosa; Resveratrol; Stilbenes; Thiazolidines; Transfection

Cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, is characterized by chronic bacterial infections and inflammation in the lung. Having previously shown that deletion of CFTR is associated with lower expression of the endogenous anti-inflammatory protein Annexin A1 (AnxA1), we investigated further this possible functional connection using a validated CFTR inhibitor. Treatment of mice with the CFTR inhibitor-172 (CFTR(172)) augmented the acute peritonitis promoted by zymosan, an effect associated with lower AnxA1 levels in peritoneal cells. Similar results were obtained with another, chemically distinct, CFTR inhibitor. The pro-inflammatory effect of CFTR(172) was lost in AnxA1(-/-), as well as CFTR(-/-) mice. Importantly, administration of hrAnxA1 and its peptido-mimetic to CFTR(-/-) animals or to animals treated with CFTR(172) corrected the exaggerated leukocyte migration seen in these animals. In vitro assays with human Polymorphonuclear leukocyte (PMN) demonstrated that CFTR(172) reduced cell-associated AnxA1 by promoting release of the protein in microparticles. We propose that the reduced impact of the counterregulatory properties of AnxA1 in CF cells contributes to the inflammatory phenotype characteristic of this disease. Thus, these findings provide an important insight into the mechanism underlying the inflammatory disease associated with CFTR inhibition while, at the same time, providing a novel pharmacological target for controlling the inflammatory phenotype of CF.

Topics: Animals; Annexin A1; Benzoates; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Peritonitis; Thiazolidines; Zymosan

The aim of this study was to characterize the role of CFTR during Cd(2+)-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr(+/+) and cftr(-/-) mice were used. In cftr(+/+) cells, the application of Cd(2+) (5 microM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl(-) conductance sensitive to CFTR(inh)-172. Thereafter Cd(2+) induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl(-) and TASK2 K(+) conductances. By contrast, cftr(-/-) cells exposed to Cd(2+) were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr(+/+) cells, Cd(2+) enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR(inh)-172 or in cftr(-/-) cells. Additionally, Cd(2+) exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr(+/+) cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd(2+) exposure by modulating the intracellular content of glutathione.

Topics: Animals; Apoptosis; Benzoates; Cadmium; Caspase 3; Cell Culture Techniques; Cell Line, Transformed; Cell Size; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Extracellular Signal-Regulated MAP Kinases; Glutathione; Kidney Tubules, Proximal; Membrane Potentials; Mice; Mice, Inbred CFTR; Patch-Clamp Techniques; Potassium Channels, Tandem Pore Domain; Reactive Oxygen Species; Signal Transduction; Thiazolidines

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Topics: Animals; Benzoates; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dogs; Epithelial Cells; Humans; Hydrogen-Ion Concentration; Lysosomes; Mice; Mice, Knockout; Respiratory Mucosa; Thiazolidines; Vacuolar Proton-Translocating ATPases

Retention of F508del-CFTR proteins in the endoplasmic reticulum (ER) is dependent upon chaperone proteins, many of which require Ca(2+) for optimal activity. Here, we show in human tracheal gland CF-KM4 cells, that after correction of F508del-CFTR trafficking by miglustat (N-butyldeoxynojirimycin) or low temperature (27 degrees C), the Ca(2+) mobilization is decreased compared to uncorrected cells and becomes identical to the Ca(2+) response observed in non-CF MM39 cells. In CF-KM4 and human nasal epithelial CF15 cells, we also show that inhibiting vesicular trafficking by nocodazole prevents not only the rescue of F508del-CFTR but also the Ca(2+) mobilization decrease. Finally, experiments using the CFTR inhibitor CFTR(inh)-172 showed that the presence but not the channel activity of F508del-CFTR at the plasma membrane is required to decrease the Ca(2+) mobilization in corrected CF cells. These findings show that correction of the abnormal trafficking of F508del-CFTR proteins might have profound consequences on cellular homeostasis such as the control of intracellular Ca(2+) level.

Topics: 1-Deoxynojirimycin; Adenosine Triphosphate; Benzoates; Boron Compounds; Calcium Signaling; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Epithelial Cells; Histamine; Humans; Inositol 1,4,5-Trisphosphate Receptors; Nocodazole; Protein Transport; Respiratory Mucosa; Sequence Deletion; Temperature; Thiazolidines

Cystic fibrosis (CF) is an autosomal recessive disease that results from mutations in the CF transmembrane conductance regulator (CFTR) gene. The effect of interventions aimed at correcting the CF electrophysiologic phenotype has been primarily measured using in vitro methods in gastrointestinal and respiratory epithelia. A reliable in vivo assay of CFTR function would be of great value in the investigation of pharmacologic interventions for CF mouse models. We performed the in vivo rectal potential difference (RPD) assay on three different mouse models. We then compared the in vivo data with the results obtained using the in vitro Ussing chamber method. The results from the in vitro method correlated closely with the results acquired using the in vivo method and were reproducible. The data suggest that the in vivo RPD assay is a reliable assay of functional CFTR expression in CF mouse models.

Topics: Animals; Benzoates; Colforsin; Colon; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Genotype; Intestinal Mucosa; Membrane Potentials; Mice; Mice, Inbred CFTR; Mice, Mutant Strains; Phenotype; Rectum; Reproducibility of Results; Thiazolidines

Cystic fibrosis (CF) is a disease produced by mutations in the CFTR channel. We have previously reported that the CFTR chloride transport activity indirectly regulates the differential expression of several genes, including SRC and MUC1. Here we report that MT-ND4, a mitochondrial gene encoding a subunit of the mitochondrial Complex I (mtCx-I), is also a CFTR-dependent gene. A reduced expression of MT-ND4 was observed in CFDE cells (derived from a CF patient) when compared to CFDE cells ectopically expressing wild-type CFTR. The differential expression of MT-ND4 in CF was confirmed by RT-PCR. In situ hybridizations of deparaffinized human lung tissue slices derived from wt-CFTR or CF patients also showed downregulation of ND4 in CF. In addition, the CFTR chloride transport inhibitors glibenclamide and CFTR(inh)-172 also reduced MT-ND4 expression in CFDE cells ectopically expressing wt CFTR. These results suggest that the CFTR chloride transport activity indirectly up-regulates MT-ND4 expression.

Topics: Base Sequence; Benzoates; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Gene Expression; Glyburide; Humans; In Situ Hybridization; Lung; Molecular Sequence Data; NADH Dehydrogenase; Thiazolidines

Airway submucosal glands have been proposed as a primary site for initiating and sustaining airway disease in cystic fibrosis (CF). However, it has been difficult to define the role of CFTR in gland fluid secretion because of concerns in interpreting experiments on diseased CF human airways subjected to chronic infection and inflammation. Here, we test the role of CFTR in gland fluid secretion by using a selective CFTR inhibitor (CFTRinh-172) in pig and human airways. Measurements of single-gland fluid secretion rates showed inhibition of both cholinergic and cAMP-stimulated fluid secretion by CFTRinh-172. Secreted fluid [Na+] and [Cl-] measured by fluorescence ratio imaging were 101 and 116 mM, respectively, and not significantly altered by secretory agonists or CFTR inhibition. Gland fluid pH was 7.1 and reduced by 0.4 units after CFTR inhibition. Gland fluid viscosity, determined by photobleaching of FITC-dextran, was threefold increased in pilocarpine-stimulated gland fluid after CFTR inhibition, and protein concentration was increased from 12 to 20 mg/ml. Our data provide strong evidence that gland fluid secretion is CFTR-dependent. The relatively hyper-viscous and acidic fluid secretions produced by acute CFTR inhibition support a role for defective gland function in CF lung disease and provide a rational basis for pharmacological creation of a large animal model of CF.

Topics: Animals; Benzoates; Body Fluids; Bronchi; Cells, Cultured; Chlorides; Cholinergic Agents; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exocrine Glands; Humans; Hydrogen-Ion Concentration; Pilocarpine; Second Messenger Systems; Sodium; Swine; Thapsigargin; Thiazoles; Thiazolidines; Viscosity