Chloride Secretion of Murine Tracheocyte Monolayers Is Stimulated by Reactive Nitrogen and Oxygen Species

Abstract 2103 Pulmonary: General Lung Biology Poster Symposium, Sunday, 5/2

Ion transport across airway epithelial cells plays an important part in the hydration of mucus and mucosal hygiene. We investigated whether reactive nitrogen and oxygen species, which may be released in inflammatory process, alter chloride transport in tracheobronchial epithelia. Tracheocytes were isolated from CD-1 mice by enzymatic digestion and cultured for 9-11 days on polycarbonate filgers on an air-liquid interface, until monolayers with a transepithelial resistance > 1000Ω*cm² were formed. The filters were then mounted in Ussing chambers (volume 6.5 ml each side), which were filled with Ringer's solution, with glucose replaced by mannitol on the apical side, bubbled with a mixture of 95% O₂ and 5% CO₂ (pH 7.4) and heated to 37°C. Sodium absorption was blocked in all experiments by adding 10 µM amiloride to the apical side, which reduced I_SC from 58±1.1 µA/cm² (mean±SEM) to 6.5±0.2 µA/cm². 3-morpholinosydnominine (SIN-1), a substance that decomposes by releasing superoxide (O₂^-) and nitric oxide (NO), which then may combine to form peroxynitrite (ONOO^-), elicited a gradual increase of I_SC over 60-90 minutes in a concentration dependent manner (ANOVA, p<0.0001), when added to both sides of the chambers. Further, SIN-1 led to a significant decrease of the chloride secretion induced by 10µM forskolin (p<0.0005). (Table)

Table 1 No caption available

The increase of I_SC by SIN-1 was almost completely abolished by glibenclamide (200 µM) and diphenylamine carboxylate (200 µM), which are known to block the cystic fibrosis transmembrane regulator (CFTR), a chloride channel. However, the increase of I_SC was not inhibited by the oxygen radical scavengers superoxide dismutase (SOD, 1000-2000 U/ml) and uric acid (200 µM), but partially inhibited by Mn-TBAP (1000-2000 U/ml), an SOD analogue. The NO donor spermine NONOate (200 µM), hydrogen peroxide (1mM), sodium nitrate (1mM) sodium nitrite (1mM) and the inactive breakdown product of SIN-1, SIN-1c did not increase I_SC. We concluded that reactive oxygen-nitrogen species increase baseline chloride across murine tracheocytes but diminish the extent of forskolin stimulation on chloride secretion. We speculate that peroxynitrite may be the major compound involved in this process.