3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone and Disease-Models--Animal

Topics: Administration, Topical; Animals; Benzoates; Cautery; Chloride Channels; Corneal Injuries; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Dry Eye Syndromes; Female; Lacrimal Apparatus; Lissamine Green Dyes; Mice; Mice, Inbred BALB C; Nanotechnology; Tears; Thiazolidines; Treatment Outcome; Triazines

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

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

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. Screening of approximately 110000 small synthetic and natural compounds for inhibition of halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure-activity relationships. The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4',5'-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chloride current with IC(50) approximately 90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date.

Topics: Animals; Cell Line; Cystic Fibrosis Transmembrane Conductance Regulator; Cysts; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Inhibitory Concentration 50; Membrane Potentials; Mice; Polycystic Kidney Diseases; Quinoxalines; Rats; Structure-Activity Relationship

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