Airway surface liquid osmolality measured using fluorophore-encapsulated liposomes

The airway surface liquid (ASL) is the thin layer of fluid coating the lumi nal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL osmolality include epithelial cell solute and water transport properti es, evaporative water loss, and the composition of secreted fluids. We deve loped a noninvasive approach to measure ASL osmolality using osmotically se nsitive 400-nm-diam liposomes composed of phosphatidylcholine/cholesterol/p olyethylene glycol-phosphatidylcholine (1:0.3:0.08 molar ratio). Calcein wa s encapsulated int he liposomes at self-quenching concentrations (30 mM) as a volume-sensitive marker, together with sulforhodamine 101 (2 mM) as a vo lume-sensitive reference. Liposome calcein/sulforhodamine 101 fluorescence ratios responded rapidly (<0.2 s) and stably to changes in solution osmolal ity. ASL osmolality was determined from calcein/sulforhodamine 101 fluoresc ence ratios after addition of microliter quantities of liposome suspensions to the ASL. In bovine airway epithelial cells cultured on porous supports at an air-liquid interface, ASL thickness (by confocal microscopy) was 22 < mu>m and osmolality was 325 +/- 12 mOsm. In anesthetized mice in which a tr ansparent window was created in the trachea, ASL thickness was 55 mum and o smolality was 330 +/- 36 mOsm. ASL osmolality was not affected by pharmacol ogical inhibition of CFTR in airway cell cultures or by genetic deletion of CFTR in knockout mice. ASL osmolality could be increased substantially to >400 mOsm by exposure of the epithelium to dry air; the data were modeled m athematically using measured rats of osmosis and evaporative water loss. Th ese results establish a ratio imaging method to map osmolality in biologica l compartments. ASL fluid is approximately isosmolar under normal physiolog ical conditions, but can become hyperosmolar when exposed to dry air, which may induce cough and airway reactivity in some patients.