Extracellular autocrine nucleotide signalling in a microenvironment: integrative physiology in a minute volume of airway surface liquid

Before the cystic fibrosis (CF) gene was cloned and the topology of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was mapped, there were multiple schools of thought and possible answers for what functional protein the CF gene encoded: dysfunctional chloride (Cl⁻) channel?; hyperactive sodium (Na⁺) channel?; and/or signal transduction effector and regulator of both Cl⁻ and Na⁺ channels? The answer was a member of the ATP-binding cassette (ABC) transporter family that acted as an anion channel and as a regulator of multiple ion, acid–base and water transport proteins and cellular processes, including functioning as an inhibitor of Na⁺ channels.

Loss of CFTR not only leads to loss of Cl⁻ permeability and transport across epithelial cell membranes but it also to up-regulation of ENaC Na⁺ channels via mechanisms that remain debated, poorly defined, and likely multifactorial. Some have suggested that CFTR may be a signal transduction effector molecule itself. Its latter regulatory and signalling effector capability may be at the root of the CF genotype and lung and airways CF disease phenotype discordance observed to date in the CF condition.

In an article in this issue of The Journal of Physiology, Button et al. (2007) have set the bar in both longevity and innovation in their quest to define the ion transport processes and their regulation within a tiny volume of extracellular fluid that bathes beating respiratory cilia, the so-called airway surface liquid (ASL) layer. Including this article, the UNC CF research group has published at least 11 original articles and reviews focused on aspects of the ASL and its regulation in the past several years (Lazarowski et al. 2004; Okada et al. 2004, 2006; Douillet et al. 2005; Tarran et al. 2005, 2006a,b; Matsui et al. 2006; Winters et al. 2006; Boucher, 2007; Button et al. 2007). Their power derives from the well-differentiated non-CF (‘normal’) and CF human airway epithelial cell monolayers that they grow in primary cultures to maintain cilia and a pseudo-stratified architecture. With this in vivo-like culture model, they have brought innovative methods as well as emerging mathematical modelling to these questions. Such a system is elegantly presented in an initial model figure by Button et al. (2007).

To be brief, their collective work in this paper and as a whole over the past 5 years is summed by the cartoon provided in Fig. 1. It is the over-arching hypothesis of this group that secreted nucleotides and nucleosides within the ASL are key to local control of anion and cation channels and to the control of the volume of the ASL. Up-regulation of ATP secretion may be critical to the normalization of ASL depth. The ASL may be one of the most tightly regulated microenvironments in the body. With these well-differentiated non-CF and CF cultures, this paper and past papers before it showed that ASL in normal physiology is maintained at a depth of 7 μm, the approximate length of the beating respiratory cilia. In CF, however, the ASL volume is depleted to more than 50% of that depth, causing a ciliary beat and mucociliary clearance problem. When different mechanical stimuli are applied to this system (phasic motion, cyclic stress, chest percussion therapy?, exercise?), one can deepen the ASL in the normal model (not shown in Fig. 1). However and importantly for future therapeutic angles for CF, CF ASL volume is restored with these mechanical stimuli, some of which simulate normal breathing and some of which are designed to perturb the system and loosen the dehydrated and tenacious mucus. They propose and show that the accumulation of secreted ATP and its metabolities (principally adenosine) reset the normal regulation of ASL volume by rescuing Cl⁻ and water secretion, while dampening Na⁺ hyperabsorption. The rescue of Cl⁻ and anion secretion is independent of CFTR, because it remains trapped within the cell. An assumption is that water channel function remains intact in CF and follows osmotic forces within the ASL and the airway surface epithelium. Indeed, secreted ATP is also critical for regulation of cell volume within the columnar airway epithelial cells as well. Aquaporin biology is difficult to study in vitro, because expression of epithelial aquaporins is often lost in cell culture for reasons unknown.

Figure 1. Cartoon depicting control of airway surface liquid via the integrative function of anion, cation and water transport and the local release of nucleotides and nucleosides with cyclic stress, phasic motion and other appropriate mechanostimuli.

Wild-type CFTR is processed normally through the ER and Golgi to the ciliated apical membrane of airway surface epithelia (left). delF508-CFTR is retained in the ER due to a folding anomaly caused by a single phenylalanine deletion in a segment of the polypeptide that connects TMD1 and NBD1. This mutation occurs in approximately 70% of affected CF patients and on 90% of CFTR alleles (centre and right). In the normal physiology, there is a balance of NaCl and water transport governed in part by CFTR that sets proper ASL depth (left). In most CF, Cl⁻ secretion is mostly lost due to retention of CFTR, and Na⁺ absorption (mainly due to ENaC) becomes hyperactive. As a result, water is absorbed too vigorously in CF due to osmotic forces. Dehydration of the airway surface liquid (centre) causes a ‘domino effect’ of problems with ciliary beat, mucociliary clearance, accumulation of dehydrated mucus and infection with opportunistic bacteria. The green and orange colour depicts non-mucoid and mucoid Pseudomonas aeruginosa bacteria accumulating within the thickened mucus in progressing CF disease. With the novel and different mechanical stimuli applied by Button, Picher, and Boucher in this and other studies, stimulation of secretion and enhancement of levels of nucleotides (ATP) and nucleosides (adenosine, Ado) inhibit ENaC-mediated Na⁺ hyperabsorption and rescue Cl⁻ secretion through ‘rescue Cl⁻ channels’ independent of CFTR that are expressed by airway and other epithelia.

Boucher and coworkers should be congratulated on this work and lauded for their comprehensive and innovative approach in this arena. This collective work is another example of how local signalling by purinergic ligands can never be overlooked as a critical cog in the minute-to-minute regulation of membrane, cell and tissue biology.