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Fluid transport across cultured rat alveolar epithelial cells: a novel in vitro system

Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130

Submitted 30 May 2003 ; accepted in final form 19 February 2004

Previous studies have used fluid-instilled lungs to measure net alveolar fluid transport in intact animal and human lungs. However, intact lung studies have two limitations: the contribution of different distal lung epithelial cells cannot be studied separately, and the surface area for fluid absorption can only be approximated. Therefore, we developed a method to measure net vectorial fluid transport in cultured rat alveolar type II cells using an air-liquid interface. The cells were seeded on 0.4-µm microporous inserts in a Transwell system. At 96 h, the transmembrane electrical resistance reached a peak level (1,530 ± 115 ·cm²) with morphological evidence of tight junctions. We measured net fluid transport by placing 150 µl of culture medium containing 0.5 µCi of ¹³¹I-albumin on the apical side of the polarized cells. Protein permeability across the cell monolayer, as measured by labeled albumin, was 1.17 ± 0.34% over 24 h. The change in concentration of ¹³¹I-albumin in the apical fluid was used to determine the net fluid transported across the monolayer over 12 and 24 h. The net basal fluid transport was 0.84 µl·cm^–2·h^–1. cAMP stimulation with forskolin and IBMX increased fluid transport by 96%. Amiloride inhibited both the basal and stimulated fluid transport. Ouabain inhibited basal fluid transport by 93%. The cultured cells retained alveolar type II-like features based on morphologic studies, including ultrastructural imaging. In conclusion, this novel in vitro system can be used to measure net vectorial fluid transport across cultured, polarized alveolar epithelial cells.

transepithelial transport; lung

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