Nitric Oxide Reduces Na-K-2Cl Cotransport in Distal Lung Epithelial Cells from Fetal Sheep • 1621

Abstract

Before birth secretion of Cl- into the lung lumen by the respiratory epithelium provides the osmotic force for transepithelial water movement and resultant filling of the fetal lung with liquid. Production of lung liquid is dependent on Na-K-2Cl cotransport, indicating that Cl- transport by this mechanism is important in fetal lung liquid secretion. After birth, Cl- secretion diminishes and Na+ transport-linked liquid reabsorption is established. The mechanism by which Cl-linked fluid secretion decreases after birth is uncertain, but we hypothesized that nitric oxide may be involved because it reduces lung liquid production in fetal lambs by a mechanism independent of Na+ reabsorption (J Appl Physiol 83: 1538, 1997). To test this notion, we studied the effect of nitric oxide on Na-K-2Cl cotransport in a line of cultured epithelial cells derived from the distal lung of a fetal lamb. After harvesting the cells into suspension, we incubated the cells for 5 min in a buffered salt solution saturated with nitrogen (control) or nitric oxide (≅6 × 10-7 M) and then calculated K+ uptake (using the K+ substitute86 Rb+) in the presence and absence of bumetanide (10-4 M) to determine the bumetanide-sensitive fraction of Rb uptake, an index of Na-K-2Cl activity. Bumetanide-sensitive Rb+ uptake averaged 58 ± 28 (nmol/million cells)/h under control conditions and was reduced (on average by 54%) to 30 ± 30 (nmol/million cells)/h when exposed to nitric oxide(n=6, mean ± SD, p < 0.002). To confirm this result, we used s-nitroso-n-acetylpenicillamine (SNAP, 250 μM) as an alternative method of generating nitric oxide. After harvesting the cells into suspension, we incubated the cells for 10 min with SNAP and then calculated Rb+ uptake in the presence and absence of bumetanide. SNAP reduced bumetanide-sensitive Rb+ uptake (on average by 47%) from 26 ± 8 (nmol/million cells)/h to 15 ± 10 (nmol/million cells)/h (control vs SNAP, n=6, p< 0.01). Thus, in fetal distal lung epithelial cells in culture, nitric oxide inhibits Na-K-2Cl cotransport, an ion transport mechanism that is important in the production of lung liquid in the fetus. We speculate that nitric oxide may play a role in reducing Cl- transport in the developing lung after birth.