(Taken directly from the application) It is unknown how in Cystic Fibrosis (CF) a """"""""simple"""""""" reduction of Cl permeability could account for preferential binding and colonization by Pseudomonas aeruginosa in CF lungs. Two hypotheses, not mutually exclusive, that could account for the pathologies are based on the fact that the CFTR Cl channel could exert its effect through alterations of pH of organelles or at the plasma membrane. The organelle pH hypothesis is that pH of trans-Golgi network (pHTGN ) and other organelles is altered due to altered Cl and HCO3 permeability of organelle membranes. Changes in pHTGN in turn lead to altered sialyltransferase activity, reduced sialylation of GM1 and increased binding of Pseudomonas aeruginosa to asialo-GM1. Alternatively, the cytoplasmic/airway pH hypothesis states that cytoplasmic pH (pHC) is elevated while epithelial apical pH (pH apical) is reduced due to reduced Cl and HCO3 exit from the cell. Elevated cytosolic pH (pHC) increases rate of vesicular transport out of Golgi/TGN, which reduces the residence time of GM1 in the secretory pathway and hence time for sialylation reactions. In addition, airway fluid will be more acidic, which may influence Pseudomonas colonization. Using molecular targeting, digital imaging microscopy and biochemical analyses, we will test the role of CFTR in control of pH apical, pHC, pHTGN, rates of TGN vesicle traffic, GM1 sialylation and Pseudomonas binding of airway epithelial cells. We will: 1. Determine role of CFTR in controlling apical fluid pH. The cytoplasmic/airway pH hypothesis predicts pH apical should be reduced in CF cells and in normal cells when CFTR is inhibited. 2. Determine role of CFTR in control of pHC (digital imaging of cytosolic, pH-sensitive dye) and pHTGN (digital imaging of pH-sensitive dyes targeted solely to the TGN) in control and in untreated and CFTR-rescued CF airway cells. The organelle pH hypothesis predicts that pHTGN of CF cells is greater than the pHTGN of CFTR-rescued cells. 3. Determine role of CFTR, pHC and pHTGN in TGN vesicular traffic, GM1 sialylation and Pseudomonas binding in untreated and CFTR-rescued CF airway cells. According to the cytoplasmic pH hypothesis, export from the TGN should be accelerated in CF compared to control or CFTR-rescued cells, and the CF phenotype should be reversed by slowing transport out of the TGN using inhibitors or by lowering pHC. The cytoplasmic/airway hypothesis also predicts that alterations of pH airway or pHC should predictably control GM1 sialylation and Pseudomonas binding. The organelle pH hypothesis predicts that sialylation of GM1 is impaired in CF and that Pseudomonas binding should be altered when pHTGN is altered.
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