Abstract

The application remains focused on the mechanisms by which the Cystic Fibrosis genotype leads to clinical abnormalities.
Aim 1 is a direct extension of recent work indicating that CFTR is a modulator of intracellular vesicle fusion.
Aims 2 and 3 concern the biophysics of the airspace surface liquid (ASL) layer. The proposed studies utilize novel optical methods including fluorescent indicators of [Cl/-] and vesicle fusion, photobleaching and time-resolved fluorescence measurements of viscosity, and 3-d tracking.
Specific Aim 1. To investigate the mechanisms and significance of the CFTR-dependent stimulation of intracellular vesicle fusion. We reported that expression of cAMP activation of CFTR in stably transfected 3T3 fibroblasts increases endosome fusion by greater than 2-fold. New technical developments will be used to determine whether CFTR regulates endosome fusion in native CFTR-expressing versus 'CF' epithelial cells and whether vesicle fusion in the secretory pathway is regulated by CFTR. The hypothesis will be tested that syntaxins are involved in the CFTR- regulation of endosome fusion.
Specific Aim 2 : To develop quantitative fluorescence methods to measure ASL fluid thickness, ionic composition and viscosity. Novel Cl/- and Na+- sensitive indicators will be used to measure ASL fluid composition in situ without fluid sampling and other perturbations. Photobleaching recovery and time-resolved fluorescence anisotrophy will be used to measure ASL fluid viscosity. Measurements will be carried out on primary cultures of human and bovine tracheal epithelia for comparison with intact bovine and monkey trachea. The role of submucosal glands in ASL fluid properties will be studied.
Specific Aim 3. To study the regulation of ASL fluid thickness, composition and viscosity, and to characterize their abnormality in Cystic Fibrosis. The methods developed in Aim 2 will be used to compare CFTR- expressing versus CF-epithelia. The hypotheses will be tested that ASL salt concentration and viscosity are elevated in CF, epithelial, and that UTP normalizes ASL fluid properties.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center (P50)
Project #
5P50HL060288-02
Application #
6202600
Study Section
Project Start
1999-09-01
Project End
2000-08-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
University of California San Francisco
Department
Type
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143

  Publications

Fischer, Horst; Illek, Beate; Finkbeiner, Walter E et al. (2007) Basolateral Cl channels in primary airway epithelial cultures. Am J Physiol Lung Cell Mol Physiol 292:L1432-43
Choi, Hyon; Fleming, Neal W; Serikov, Vladimir B (2007) Contact activation via ICAM-1 induces changes in airway epithelial permeability in vitro. Immunol Invest 36:59-72
Dix, James A; Hom, Erik F Y; Verkman, A S (2006) Fluorescence correlation spectroscopy simulations of photophysical phenomena and molecular interactions: a molecular dynamics/monte carlo approach. J Phys Chem B 110:1896-906
Sonawane, N D; Muanprasat, Chatchai; Nagatani Jr, Ray et al. (2005) In vivo pharmacology and antidiarrheal efficacy of a thiazolidinone CFTR inhibitor in rodents. J Pharm Sci 94:134-43
Oshio, Kotaro; Watanabe, Hiroyuki; Song, Yaunlin et al. (2005) Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1. FASEB J 19:76-8
Hara-Chikuma, Mariko; Verkman, A S (2005) Aquaporin-3 functions as a glycerol transporter in mammalian skin. Biol Cell 97:479-86
Thiagarajah, Jay R; Broadbent, Talmage; Hsieh, Emily et al. (2004) Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor. Gastroenterology 126:511-9
Schwarzer, Christian; Machen, Terry E; Illek, Beate et al. (2004) NADPH oxidase-dependent acid production in airway epithelial cells. J Biol Chem 279:36454-61
Serikov, Vladimir B; Choi, Hyon; Chmiel, Ken J et al. (2004) Activation of extracellular regulated kinases is required for the increase in airway epithelial permeability during leukocyte transmigration. Am J Respir Cell Mol Biol 30:261-70
Irokawa, Toshiya; Krouse, Mauri E; Joo, Nam Soo et al. (2004) A ""virtual gland"" method for quantifying epithelial fluid secretion. Am J Physiol Lung Cell Mol Physiol 287:L784-93

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