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A7620 - Intragranular Mucin Packaging in Normal and Cystic Fibrosis Human Bronchial Epithelial Cells
Author Block: O. Ponomarchuk1, S. Raquena2, R. Fudala2, I. Gryczynski2, S. Dzyuba3, Z. Gryczynski4, E. Brochiero5, R. Grygorczyk5; 1Centre de recherche du CHUM, Montreal, QC, Canada, Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russian Federation, 2University of North Texas Health Science Center, Center for Cancer Research, Institute for Molecular Medicine, Fort Worth, TX, United States, 3Department of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX, United States, 4Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, United States, 5Department of Medicine, Université de Montréal, Centre de recherche du CHUM, Montreal, QC, Canada.
RATIONALE: Airway mucus is formed by secretion and hydration of gel-forming polymeric mucin macromolecules (MUC5AC, MUC5B). Mucins are synthesized and stored in a highly-condensed form inside intracellular granules of secretory epithelial cells, and they are then released from granules through exocytosis. Swelling process and viscosity of released mucus are critically affected by extracellular airway surface liquid (ASL) pH and Ca2+ concentration, both of which are modulated by CFTR-dependent HCO3- secretion. In Cystic Fibrosis (CF), defective CFTR function leads to abnormally viscous and sticky mucus, resulting in a variety of pathological conditions. While acidic ASL pH may explain some of CF mucus abnormalities, the existence and contribution of defects intrinsic to the stored granules, such as alkaline intragranular pH and/or altered mucin packaging, remain unclear and constitute the focus of our study.
METHODS: We used a noninvasive approach that includes Fluorescence Life Time Imaging Microscopy (FLIM) and fluorescent BODIPY-based molecular viscometers to assess intragranular mucin matrix viscosity of non-CF and CF (ΔF508 defect) Human Bronchial Epithelial cells. Effects of rising pH inside granules (10 nM Bafilomycin A, BFA) on intragranular mucin viscosity or effects of reducing agent (1 mM DTT) were investigated.
RESULTS: In mucus-producing CF cells, a subpopulation of mucin granules with a significantly reduced viscosity of the intragranular matrix was found when compared to control cells from healthy patients. At the same time, the size distribution of the mucin granules was not different in CF and in non-CF cells. Preliminary data showed that treatment of non-CF cells with BFA significantly affected intragranular mucin’s viscosity, making their properties similar to those observed in CF cells.
CONCLUSIONS: Lower viscosity may suggest abnormal packaging/condensation of mucin macromolecules in CF granules that may impact their release and swelling process. Our data suggest that clinically observed abnormalities in CF mucus may be already reflected in the mucin properties stored in granules prior to their secretion.