EXCEED THE SPACE PROVIDED. Increasingevidence suggests that the pH of the airway surface liquid(pHAsL)plays an important role in inflammatory airway diseases, such as cystic fibrosisand asthma. Normal pHAsLis slightlyacidic (pH = 6.9) compared to plasma (pH = 7.4). The source of the acidityis not known. In our preliminary data we show that human airway surface epithelia secrete acid by way of an apical membrane H Acid secretion is regulated by mucosal histamine and ATP, two mediators that are released in vivo during airway inflammation and stress. In studies on a variety of airway epithelialcell cultures, H* secretion was only present in those cultures that expressed mRNA for the putative proton channel NADPH oxidase homolog 4 (NOX4). Therefore, based on these preliminary data we hypothesize that acidificationof the airway surface liquidis caused by a NOX4 proton conductance. This hypothesiswill be tested in three ways.
In Specific Aim 1 we will determine the properties of the native H* conductance and the underlyingH* channels expressed in airway surface epithelia using whole cell patch clamping, noise analysis, and pH stat recordings. The regulation of the H by the membrane voltage, intra- and extracellular pH, and its sensitivityto blockers will be studied. Physiological drivingforces for H* exit across the apical membrane will be measured using microelectrodes and confocal imaging.The connection between H* secretion and superoxide generation by NADPH oxidase will be investigated.
Specific Aim 2 is to determine the sequence and molecular expression of NOX4 in human airways. We will clone full-length NOX4 cDNA from human airway surface cells. Plasma membrane localizationof NOX4 will be determined using a NOX4-GFP fusion protein. Cell type-specific expression of NOX4 will be determined by in situ hybridization of tracheal sections.
Specific Aim 3 is to identifythe functionof NOX4 as the H* channel in airway epithelia. The biophysicaland physiologicalparameters of the recombinant NOX4 H* conductance will be determined and quantitatively compared to the native H* conductance. Antisense probes for NOX4 will be used to selectively inhibitexpressionof a H* conductance in airway cells expressing endogenous NOX4. The resultsof our studieswill reveal the molecular, physiologicaland biophysical properties of the native H* conductance and NOX4 in airways. In the longterm, this research will improve our understandingof the mechanism responsible for acidificationof the airway surface liquid.This will help develop novel therapeuticaltargets for the treatment of airway acidity and inflammatory airway diseases. PERFORMANCE SITE ========================================Section End===========================================
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