Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia.

A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-mum periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to >or=7 microm are poorly understood. Using bumetanide as an inhibitor of Cl- secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated "blending" of both Cl- secretion and Na+ absorption must occur to effect ASL volume homeostasis. We then investigated how ASL volume status is regulated by the underlying epithelia. Cilia were not critical to this process as (a) ASL volume was normal in cultures from patients with primary ciliary dyskinesia with immotile cilia, and (b) in normal cultures that had not yet undergone ciliogenesis. However, we found that maneuvers that mimic deposition of excess ASL onto the proximal airways, which occurs during mucociliary clearance and after glandular secretion, acutely stimulated Na+ absorption, suggesting that volume regulation was sensitive to changes in concentrations of soluble mediators in the ASL rather than alterations in ciliary beating. To investigate this hypothesis further, we added potential "soluble mediators" to the ASL. ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl- secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume.

Ionic mechanism of forskolin-induced liquid secretion by porcine bronchi.

cAMP-elevating agents such as forskolin and vasoactive intestinal peptide induce liquid secretion by tracheobronchial submucosal glands. This pathway is thought to be CFTR dependent and thus defective in cystic fibrosis; however, the ionic mechanism that drives this secretion process is incompletely understood. To better define this mechanism, we studied the effects of ion transport inhibitors on the forskolin-induced liquid secretion response (Jv) of porcine distal bronchi. The forskolin-induced Jv was driven by a combination of bumetanide-sensitive Cl- secretion and DIDS-sensitive HCO3- secretion. When Cl- secretion was inhibited with bumetanide, Na+/H+ exchange-dependent HCO3- secretion was apparently induced to compensate for the loss of Cl- secretion. The forskolin-induced Jv was significantly inhibited by the anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, and glibenclamide. We conclude that the forskolin-induced Jv shares many characteristics of cholinergically induced secretion except for the presence of a DIDS-sensitive component. Although the identity of the DIDS-sensitive component is unclear, we speculate that it represents a basolateral membrane Na+ -HCO3- cotransporter or an Na+-dependent anion exchanger, which could account for transepithelial HCO3- secretion.

Disruptive effects of anion secretion inhibitors on airway mucus morphology in isolated perfused pig lung.

Since anion secretion inhibitors reproduce important aspects of cystic fibrosis (CF) lung disease, the effects of these antagonists on airway mucus morphology were assessed in isolated perfused pig lungs. Maximal inhibitory concentrations of bumetanide and dimethylamiloride, which respectively block Cl- and HCO3- secretion in porcine airways, induced the formation of dense 'plastered' mucus on the airway surface, depletion of periciliary fluid and collapse of cilia. This effect was more pronounced when lungs were also exposed to bethanechol to stimulate submucosal gland secretion, when plastered mucus covered > 98 % of the airway surface. Bethanechol also reduced gland duct mucin content in the absence, but not presence, of the anion secretion inhibitors. Anion secretion inhibitors did not induce measurable increases in goblet cell degranulation. We conclude that inhibition of anion and liquid secretion in porcine lungs disrupts the normal morphology of airway surface mucus, providing further evidence that impaired anion secretion alone could account for critical aspects of CF lung disease.

Liquid secretion inhibitors reduce mucociliary transport in glandular airways.

Because of its possible importance in cystic fibrosis (CF) pulmonary pathogenesis, the effect of anion and liquid secretion inhibitors on airway mucociliary transport was examined. When excised porcine tracheas were treated with ACh to induce gland liquid secretion, the rate of mucociliary transport was increased nearly threefold from 2.5 +/- 0.5 to 6.8 +/- 0.8 mm/min. Pretreatment with both bumetanide and dimethylamiloride (DMA), to respectively inhibit Cl(-) and HCO secretion, significantly reduced mucociliary transport in the presence of ACh by 92%. Pretreatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid similarly reduced mucociliary transport in ACh-treated airways by 97%. These agents did not, however, reduce ciliary beat frequency. Luminal application of benzamil to block liquid absorption significantly attenuated the inhibitory effects of bumetanide and DMA on mucociliary transport. We conclude that anion and liquid secretion is essential for normal mucociliary transport in glandular airways. Because the CF transmembrane conductance regulator protein likely mediates Cl(-), HCO, and liquid secretion in normal glands, we speculate that impairment of gland liquid secretion significantly contributes to defective mucociliary transport in CF.