Fluid movement between airspace and vascular compartments is important in normal lung function and pulmonary edema. This proposal is focused on biophysical (aim 1) and molecular (aim 2) water transporting mechanisms, and the role of molecular water channels in lung physiology (aim 3). The proposal consists of three specific aims:
In Specific Aim 1 they will apply novel optical methods to measure water permeability in intact lung epithelia, endothelia and individual cell membranes. Optical methods were developed in their laboratories to measure: a) airspace-to-capillary osmotic water permeability (Pf) in intact lung; b) transepithelial Pf in airways; and c) membrane Pf in epithelial layers. New optical approaches will be applied to quantify the role of epithelial vs. endothelial barriers in airspace-to-capillary water movement, of type I vs. type II cells in transalveolar water permeability, and of apical vs. basolateral Pf in airway and alveolar epithelia.
In Specific Aim 2 they will identify and characterize molecular water channels (aquaporins) that confer high water permeability in lung. Four aquaporins are expressed in lung: AQP3 (trachea) and AQP4 (airway basolateral membrane), cloned by the principal investigator, AQP1 (endothelium) and AQP5 (alveolar apical membrane) cloned by Dr. Agre. Water channels have not been identified at the alveolar basolateral membrane and airway apical membrane. The following cloning strategies will be used to identify these proteins: PCR-homology cloning using mRNA from alveolar and airway cells, and chromosome-specific exon amplification. Water channels will be studied by Xenopus oocyte expression and immunocytochemistry.
In Specific Aim 3 they will define the role of water channels in lung physiology using transgenic knock-out mice. The investigators recently generated the first animal knock-out models for water channels AQP4 and AQP1 deficient mice. Homozygous transgenic knock-out mice will be generated for AQP3 and AQP5, and characterized in terms of gross phenotype, lung water permeability, clearance of alveolar and interstitial edema, and airway resistance and reactivity.
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