Ion and fluid transport properties of small airways in cystic fibrosis.

RATIONALE: Small airways constitute a major site of pathology in cystic fibrosis (CF) and provide most of the surface area of the conducting airways of the lung. Little is known, however, about the impact of CF on ion and fluid transport in small (bronchiolar) airways. OBJECTIVES: To describe the ion and fluid transport properties of CF bronchiolar epithelium. METHODS: Primary cultures of human bronchial and bronchiolar (non-CF and CF) epithelial cells were obtained. The bioelectric properties were studied in Ussing chambers and the airway surface liquid (ASL) height was measured with confocal microscopy. MAIN RESULTS: Primary cultures of DeltaF508 CF bronchiolar epithelial cells displayed higher transepithelial resistance than non-CF cultures, whereas baseline short circuit current and amiloride-inhibitable short circuit current were similar in both preparations. The ASL height was significantly smaller in CF compared with non-CF preparations. In the presence of amiloride, addition of forskolin increased short circuit current in non-CF but not in CF bronchiolar cultures, and the ATP-induced increase in short circuit current was lower in CF than in non-CF cultures. Non-CF bronchiolar preparations displayed larger short circuit current and fluid secretion in responses to forskolin than non-CF bronchial preparations, suggesting that CFTR-dependent Cl(-) transport may play a more important role in the regulation of fluid transport in small airways than in large airways. CONCLUSION: In CF small airways, defective Cl(-) secretion combined with unregulated (persistent) Na(+) absorption results in ASLdepletion.

Isolation and air-liquid interface culture of human large airway and bronchiolar epithelial cells.

This article describes the techniques of isolation and culture of human airway epithelial cells from large airways and from distal airways. Both cell types are obtained from lung pieces collected during surgery. The protocols start with an initial step of washing and dissection of the lung pieces to separate large airways or distal airways from the surrounding parenchyma. The second step is enzymatic isolation of epithelial cells from the dissected large or distal airways. Cells are then collected by centrifugation and then seeded onto collagen surfaces. Epithelial cells can be grown at an air-liquid interface and usually form a confluent and functional epithelial layer within days.

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro.

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca2+ (Fura2) and Na+ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o- after exposure to H2O2 or *OH (H2O2 + Fe2+). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H2O2 or *OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl-. Cai also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Nai on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, *OH inhibited the Na+ current (a measure of Na(+)-K(+)-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na+/K(+)-pump may contribute to the sustained elevation of Nai. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.

[Genetics and cellular biology of cystic fibrosis]. / Génétique et biologie cellulaire de la mucoviscidose.

Cystic fibrosis (CF) is the most frequent lethal genetic disease in caucasians. It behaves as an autosomal recessive disease. The CF gene encodes a protein named CFTR (for "Cystic Fibrosis Transmembrane conductance Regulator") which exerts multiple functions. One of the most important function is the regulation of sodium and chlorine transport in epithelia. Defects in this control induce dehydration of airway surface liquid, which results in recurring bronchopulmonary infections, and increases in Na and Cl concentrations in sweat, which constitutes the rationale for the sweat test. CFTR also participates in the regulation of inflammation. Over one thousand mutations in the CFTR gene have been reported, the most frequent of which is the absence of the phenylalanine residue at amino acid position 508 (delta F508).

Effects of endothelin-1 on epithelial ion transport in human airways.

Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10(-7) M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10(-7) M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1-induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1-induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.

Characterization of ion and fluid transport in human bronchioles.

The regulation of the volume and composition of airway surface liquid is achieved through epithelial ion transport processes. In humans, these processes have been characterized in proximal but not distal airways. Segments of human bronchioles were dissected from surgically removed lung pieces. The transmural potential difference of microperfused bronchioles was inhibited by luminal exposure to amiloride and increased when exposed to the Cl secretagogues forskolin and ATP in the presence of amiloride. Human bronchiolar epithelial cells were cultured on permeable supports and studied in Ussing chambers. They generated a short circuit current (Isc) that decreased in response to amiloride and increased in response to forskolin and to ATP in the presence of amiloride. In low-Cl Kreb's Ringer bicarbonate, the baseline Isc and amiloride-induced decrease in Isc were not different, whereas the forskolin- and ATP-induced increases in Isc were smaller. Fluid transport measurement in excised bronchioles revealed a basal absorptive flow that was reduced by amiloride, whereas forskolin and ATP combined induced a secretory flow in the presence of amiloride. We conclude that human bronchioles actively absorb Na and fluid in unstimulated conditions and are capable of active Cl and fluid secretion when exposed to forskolin and to ATP.