The cystic fibrosis trans-membrane conductance regulator (CFTR), a 1480 amino acid protein, is a member of the traffic ATPase family (60) and functions as a cAMP-regulated CI channel. Based on our published results and preliminary data, we hypothesize that chronic exposure of mice and airway cells to agents which increase concentrations of reactive species (RONS), formed by the interaction of nitric oxide (NO) with partially reduced oxygen intermediates, results in oxidative modifications (oxidation, nitration and/or nitrosation) of key CFTR amino acids. These changes may: (1) decrease apical levels of CFTR by targeting it for ubiquitination and endoplasmic reticulum associated degradation by proteasomes and (2) impair Cl- secretion across the airway and alveolar epithelial cells following cAMP-stimulation by decreasing CFTR phosphorylation. These hypotheses will be tested both in vitro, by exposing Calu-3, primary human airway epithelial cells and mouse tracheocytes (MTE) to NO and RONS, as well as C57BL/6 mice to NO (1-10 ppm); nitrogen dioxide (NO2:1-10 ppm); intratracheal instillation of Mycoplasma pulmonis and measure the extent of oxidative modification and ubiquitination of CFTR as well as microscopic (single channel Cl currents) and macroscopic (whole cell Cl currents, nasal potential differences and alveolar fluid clearance) indices of its ability to act as a cAMP-activated Cl- channel. To identify specific amino acids modifications leading to loss of CFTR function, we will construct CFTR mutants by substituting each of the 40 CFTR tyrosines with alanine, express each cRNA in oocytes, and measure basal and cAMP-activated whole cell and single channel Cl- currents before and after exposure of oocyte to the RONS. Because of the well demonstrated vital importance of CFTR in both the hydration of airway fluid, as well as in cAMP-activated Na+ transport across the alveolar epithelium, the results of these studies may offer significant new insight into the pathophysiology of a number of pulmonary, non cystic fibrosis inflammatory diseases such as asthma, chronic obstructive lung disease and adult respiratory distress syndrome.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL075540-04
Application #
7325704
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Banks-Schlegel, Susan P
Project Start
2004-12-15
Project End
2009-11-30
Budget Start
2007-12-01
Budget End
2009-11-30
Support Year
4
Fiscal Year
2008
Total Cost
$344,900
Indirect Cost
Name
University of Alabama Birmingham
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Jurkuvenaite, Asta; Chen, Lan; Bartoszewski, Rafal et al. (2010) Functional stability of rescued delta F508 cystic fibrosis transmembrane conductance regulator in airway epithelial cells. Am J Respir Cell Mol Biol 42:363-72
Vitturi, Dario A; Teng, Xinjun; Toledo, José C et al. (2009) Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation. Am J Physiol Heart Circ Physiol 296:H1398-407
Matalon, Sadis; Shrestha, Kedar; Kirk, Marion et al. (2009) Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo. FASEB J 23:1415-30
Chen, Lan; Bosworth, Charles A; Pico, Tristant et al. (2008) DETANO and nitrated lipids increase chloride secretion across lung airway cells. Am J Respir Cell Mol Biol 39:150-62
Varga, Karoly; Goldstein, Rebecca F; Jurkuvenaite, Asta et al. (2008) Enhanced cell-surface stability of rescued DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones. Biochem J 410:555-64
Song, Weifeng; Matalon, Sadis (2007) Modulation of alveolar fluid clearance by reactive oxygen-nitrogen intermediates. Am J Physiol Lung Cell Mol Physiol 293:L855-8
Chen, Lan; Patel, Rakesh P; Teng, Xinjun et al. (2006) Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione. J Biol Chem 281:9190-9
Ji, Hong-Long; Su, Xue-Feng; Kedar, Shrestha et al. (2006) Delta-subunit confers novel biophysical features to alpha beta gamma-human epithelial sodium channel (ENaC) via a physical interaction. J Biol Chem 281:8233-41
Kotamraju, Srigiridhar; Matalon, Sadis; Matsunaga, Toshiyuki et al. (2006) Upregulation of immunoproteasomes by nitric oxide: potential antioxidative mechanism in endothelial cells. Free Radic Biol Med 40:1034-44