betadex and Cystic-Fibrosis

Pseudomonas aeruginosa secretes outer-membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical membrane of airway epithelial cells and decrease wt-CFTR Cl

Topics: 2-Hydroxypropyl-beta-cyclodextrin; beta-Cyclodextrins; Biofilms; Bronchi; Cell Line; Cell-Derived Microparticles; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Membrane Microdomains; Pseudomonas aeruginosa

Airway surfactant is impaired in cystic fibrosis (CF) and associated with declines in pulmonary function. We hypothesized that surfactant dysfunction in CF is due to an excess of cholesterol with an interaction with oxidation.. Surfactant was extracted from bronchial lavage fluid from children with CF and surface tension, and lipid content, inflammatory cells and microbial flora were determined. Dysfunctional surfactant samples were re-tested with a lipid-sequestering agent, methyl-β-cyclodextrin (MβCD).. CF surfactant samples were unable to sustain a normal low surface tension. MβCD restored surfactant function in a majority of samples.Mechanistic studies showed that the dysfunction was due to a combination of elevated cholesterol and an interaction with oxidized phospholipids and their pro-inflammatory hydrolysis products.. We confirm that CF patients have impaired airway surfactant function which could be restored with MβCD. These findings have implications for improving lung function and mitigating inflammation in patients with CF.

Topics: beta-Cyclodextrins; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child; Child, Preschool; Cholesterol; Cystic Fibrosis; Female; Humans; Lung Diseases, Interstitial; Male; Oxidation-Reduction; Respiratory Function Tests; Surface Properties

Changes in the level of membrane cholesterol regulate a variety of signaling processes including those mediated by acylated signaling molecules that localize to lipid rafts. Recently several types of ion channels have been shown to have cholesterol-dependent activity and to localize to lipid rafts. In this study, we have investigated the role of cholesterol in the regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, activated transepithelial short circuit current (Isc), but only from the basolateral side. Similar results were obtained with a cholesterol-binding agent, filipin, and with the sphingomyelin-degrading enzyme, sphingomyelinase. Experiments with DeltaF508CFTR mutant mice indicated that raft disruption affected CFTR-mediated anion secretion, while pharmacological studies showed that this effect was due to activation of basolateral large conductance Ca2+-activated K+ (BK) channels. Sucrose density gradient centrifugation studies demonstrated that BK channels were normally present in the high-density fraction containing the detergent-insoluble cytoskeleton, and that following treatment with MbetaCD, BK channels redistributed into detergent-soluble fractions. Our evidence therefore implicates novel high-density cholesterol-enriched plasma membrane microdomains in the modulation of BK channel activation and anion secretion in colonic epithelia.

Topics: Amiloride; Animals; beta-Cyclodextrins; Cell Membrane; Cell Membrane Permeability; Cholesterol; Colon; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Filipin; Membrane Microdomains; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Ouabain; Patch-Clamp Techniques; Potassium Channels; Quaternary Ammonium Compounds; Water