Pleural surface fluorescence measurement of Na+and Cl− transport across the air space-capillary barrier

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Abstract
We developed a pleural surface fluorescence method to measure Na+ and Cl−transport in perfused mouse lungs. The air space was filled with aqueous fluid containing membrane-impermeant fluorescent indicators of Cl− (lucigenin) or Na+ (Sodium Green). After instillation of a Cl−-free solution into the air space, an increase in perfusate Cl− concentration from 0 to 30 mM produced a decrease in surface lucigenin fluorescence (6.5%/min) corresponding to Cl− influx of 1.0 mM/min. Cl− influx was increased to 2.1 ± 0.3 mM/min by forskolin, and the increase was inhibited by glibenclamide. cAMP-stimulated Cl− influx was decreased by 57% in CFTR null mice. After instillation of a Na+-free solution into the air space, an increase in perfusate Na+ concentration from 0 to 30 mM gave increased Sodium Green fluorescence (Na+ influx of 1.2 mM/min), which increased approximately fivefold after cAMP agonists. Cl− and Na+transport were not affected in lungs from mice lacking aquaporins AQP1 or AQP5. Our results establish a pleural surface fluorescence method to measure unidirectional Cl− and Na+ flux in intact lung and provide evidence for cAMP-stimulated transcellular Cl− and Na+ transport.