Published ahead of print on December 23, 2004, doi:10.1164/rccm.200310-1423OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200310-1423OC
The Effect of Varying Tonicity on Nasal Epithelial Ion Transport in Cystic FibrosisDepartment of Gene Therapy, Faculty of Medicine, Imperial College at the National Heart and Lung Institute, London, United Kingdom Correspondence and requests for reprints should be addressed to Professor Eric Alton M.D., F.R.C.P., Department of Gene Therapy, Faculty of Medicine, Imperial College at the National Heart and Lung Institute, London SW3 6LR, UK. E-mail: e.alton{at}imperial.ac.uk
There is reasonable evidence that the fluid layer of the airway epithelium is exposed to changes in tonicity. The inspiration of cool, dry air causes an increased tonicity, whereas this tonicity may be decreased by glandular secretions. We hypothesized that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the responses to changes in tonicity and that these may be altered in cystic fibrosis (CF). Using nasal potential difference (PD) protocols in 8 subjects with CF and 10 subjects without CF, we investigated the effects of hyper- and hypotonicity on ion transport processes. We found significant differences between the two groups. In response to a hypertonic challenge (mannitol 500 mM), there was a decreased PD in both groups, suggesting decreased sodium absorption. However, after the prior inhibition of sodium transport using amiloride, there was an increased PD in the non-CF group alone, suggesting CFTR-mediated chloride secretion in response to luminal hypertonicity. For the hypotonic solution, we found that hypotonicity inhibited CFTR-mediated chloride secretion in the non-CF group. These data suggest that CFTR plays a role in the recognition and regulation of airway fluid tonicity.
Key Words: cystic fibrosis • cystic fibrosis transmembrane conductance regulator • osmolar concentration • transmembrane potentials Cystic fibrosis (CF) lung disease occurs as a consequence of the loss or dysfunction of the CF transmembrane conductance regulator protein (CFTR). However, there is currently no consensus explaining how the abnormalities seen in CF airway epithelial cells, namely a defective cAMP-mediated chloride secretion (1) and hyperabsorption of sodium (2) across the apical membrane, lead to abnormalities in the homeostasis of the airway surface liquid (ASL) layer and consequent failure to maintain an adequate lung defense. Accurate in vivo sampling is hampered by the fact that this tiny layer of fluid is altered by even the least invasive sampling technique (3, 4), and in vitro model systems have failed to yield consistently similar results (5–7). There are conflicting hypotheses for the pathogenesis of CF lung disease in relation to a dysfunction in ion transport processes (8). One suggests that there is an increased absorption of an isotonic ASL in CF, leading to ineffective mucociliary clearance (5). Another suggests that the underlying composition of the ASL is altered in CF, with a higher concentration of chloride ions leading to a reduction in the efficacy of natural antimicrobials (7, 9). Both agree that the regulation of ASL is disturbed in the basal state. Various lines of evidence suggest that the ASL is in dynamic equilibrium, being normally subject to alterations in tonicity (10–12). The inspiration of cold, dry air causing evaporative water loss has been shown to render the ASL of both proximal (12) and distal (11) airways relatively hypertonic (10). Exercise would therefore be expected to render the ASL relatively hypertonic. In two in vivo studies, exercise was shown to depolarize the potential difference (PD) of those with CF (13, 14). In the former study, subjects without CF demonstrated a relative hyperpolarization (13), suggesting a role for CFTR in these fluctuations. Furthermore, it has been demonstrated that the stimulation of submucosal glands results in the secretion of a relatively hypotonic liquid (3), theoretically balancing evaporative losses. CFTR is highly expressed in the serous cells of submucosal glands (15) and is therefore well placed to play a dynamic role in ASL regulation (16). CFTR is apically localized (17) and has been shown to be able to sense changes in the external Cl– concentration (18). In an in vitro study, human nasal epithelium was shown to function as an osmotic sensor (19), with an increased luminal, but not serosal, osmolarity altering ion transport processes. Furthermore, in a patch-clamp study using human CF tracheal epithelial cell lines (20), CFTR was implicated in defective regulatory volume decrease secondary to a calcium-dependent potassium channel. The effect of changes in ASL tonicity on ion transport mechanisms has not previously been assessed in vivo despite the various lines of evidence described previously suggesting a possible role for CFTR. The ciliated epithelium of the lower turbinate of the nose is functionally similar to lower airways and nasal PD responses correlate with measurements made in the lower airways (19). Nasal PD protocols therefore represent a surrogate assay for the in vivo investigation of epithelial ion transport. We assessed two hypertonic protocols (in the presence and absence of the sodium channel inhibitor, amiloride) and two further protocols designed to investigate the effects of hypotonicity. These were compared with a well-established isotonic protocol (21), which separately assesses apical sodium and chloride channel activity and is widely used in the discrimination between subjects with and without CF. The data are consistent with a role for CFTR in the recognition and regulation of ASL tonicity. Some of the results of these studies have been previously reported in the form of an abstract (22).
Subjects Eight patients who met accepted diagnostic criteria for CF (sweat test and phenotype), who were older than 16 years, and who had a confirmed CF  F508-F508 genotype and 10 healthy, nonsmoking, subjects without CF gave written, informed consent for the study, which was approved by the ethics committee of the Royal Brompton Hospital. All subjects were free from any upper respiratory symptoms during testing and had no prior history of nasal polyps.
Nasal PD Measurements
Solutions The effect of an increased tonicity was assessed by the addition of the nonionic osmolyte, mannitol. Mannitol (500 mM) was added to the HEPES (total osmolarity 801 mOsm), both with and without amiloride pretreatment, in separate protocols. In a prior pilot study in three individuals without CF, switching the hypertonic perfusate back to the isotonic HEPES solution resulted in the return of the PD to its former baseline. Further details are available in the online supplement. Any reduction in the tonicity of HEPES will also reduce the Cl– concentration and therefore have a potentially confounding effect on the PD response. Therefore, in a pilot study, six subjects without CF underwent separate isotonic protocols containing varying Cl– concentrations to assess the magnitude of Cl– secretion each induced. Additional details on the methods used to develop these protocols are provided in an online supplement. With this knowledge, the effects of hypotonicity were assessed by comparing the responses of an isotonic to a hypotonic solution, both of which contained a constant chloride concentration (40 mM). The hypotonic solution consisted of the following: amiloride 100 µM, 10 mM HEPES, 40 mM Cl–, 24 mM Na+, 6 mM K+, 2 mM Ca2+, 1 mM Mg2+, and 10 mM glucose in deionized water (osmolarity 93 mOsm). The isotonic 40-mM chloride solution was made to isotonicity (293 mOsm) by the addition of mannitol. These protocols were performed at intervals of not less than 24 hours.
Drugs
Statistical Analysis
Baseline Values and Standard Protocol Baseline PD values and subsequent perfusion of the standard protocol clearly discriminated subjects with CF, whose PD responses were consistent with the typically increased Na+ absorption (CF –37.8 ± 2.8 mV, non-CF –17.2 ± 0.76 mV; p < 0.001) and failure of cAMP-mediated Cl– secretion (CF –19.7 ± 1.3 mV, non-CF +1.4 ± 2.1 mV; p < 0.001).
CFTR Is Involved in the Response to Hypertonicity
After amiloride pretreatment, in the non-CF group, mannitol now produced a hyperpolarization ( PD – 3.7 ± 0.9 mV). In contrast, in the CF group, there was a depolarization (PD 3.0 ± 0.5 mV). These responses to hypertonicity in the presence of amiloride differed significantly between the two groups (p < 0.01, CF vs. non-CF; Figure 2).
CFTR Is Involved in the Response to Hypotonicity After amiloride perfusion, the effects of a hypotonic solution containing 40 mM Cl– were compared with an isotonic solution with the same Cl– concentration. In subjects without CF, perfusion of the isotonic 40 mM Cl– solution resulted in a relative hyperpolarization ( PD – 6.7 ± 0.7 mV), as expected. In contrast, the hypotonic 40 mM Cl– solution caused a brief hyperpolarization but eventual relative depolarization after 5 minutes (PD 2.7 ± 0.7 mV; p < 0.001 isotonic vs. hypotonic; Figure 3A). In the CF group, the expected failure of Cl– conductance was seen in response to the isotonic 40 mM Cl– solution (PD – 1.2 ± 1.1 mV). The hypotonic 40 mM Cl– solution also demonstrated no significant difference in response (PD 0.8 mV ± 1.5) at 5 minutes (Figure 3B). Taken together, these data indicate that hypotonicity acutely inhibits CFTR-mediated chloride secretion.
Current theories attempting to explain the pathogenesis of lung disease in CF revolve around the basic electrophysiologic defect leading to an alteration in either the composition or volume in the ASL layer (8). Both hypotheses are likely to involve CFTR responding to its external environment. Experimental evidence that CFTR can function as a chloride sensor has previously been proposed (18), and it is likely that alterations in ASL chloride concentration occur secondary to changes in tonicity. Our data suggest that CFTR is involved in the response to both hypotonic and hypertonic stimuli independent of changes in Cl– concentration. The effect of hypertonicity was assessed by using an increase of 500 mOsm. The maximum recorded osmolality in the lower airways has been approximately 450 mOsm. However, in vivo sampling methods lead to the stimulation of a relatively hypotonic glandular secretion (3). Furthermore, nasal epithelial secretions are not activated until the osmolarity of a test solution is increased by much greater amounts (> 6 x normal saline) (23). In both subjects with and without CF, the hypertonic challenge resulted in a relative depolarization. However, after apical sodium entry had been inhibited by amiloride, a relative hyperpolarization was seen in subjects without CF, whereas subjects with CF showed depolarization of the nasal PD. One interpretation of these data is that luminal hypertonicity inhibits sodium absorption in both subjects with and without CF. However, if already depolarized by amiloride, hypertonicity stimulates chloride secretion. The failure in response in the CF group suggests that this effect is mediated through, or regulated by, CFTR. Unfortunately, it is unclear what change in in vivo PD is physiologically relevant. Thus, it is possible that very small changes in PD, which may be induced by more physiologically relevant tonicity but would have been harder to detect, may still be important. Pilot studies established the conditions under which a robust signal of Cl– secretion could be measured in subjects without CF, at a Cl– concentration commensurate with a hypotonic solution. By keeping the subsequent hypotonic challenge at this constant Cl– concentration, we were able to separate the potential confounding effect of a "low-chloride" solution from that of a hypotonic challenge. An isotonic solution containing 40 mM Cl– caused a relative hyperpolarization in the non-CF group but not in the CF group, consistent with the widely reported failure of apical Cl– conductance in CF. However, when the tonicity of this solution alone was reduced to 100 mOsm, the non-CF response was inhibited, suggesting that CFTR is also involved in the response to hypotonicity. Lower ASL tonicity could occur after submucosal gland secretion. Given the complex and poorly understood nature of the regulation of the ASL, one advantage of in vivo testing is the presence of an intact epithelium, which may undergo physiologically relevant processes. However, there are recognized limitations to this method of study. Most notably, changes in PD reflect changes across the whole epithelium, namely the apical and basolateral membranes and the paracellular pathway. It is not therefore possible to categorically implicate a single ion channel in these measurements. However, because the difference between the two groups studied is the presence or absence of functional CFTR, we propose the involvement of CFTR either directly or via its interaction with other ion channels to account for the findings described previously. We suggest that the changes in PD likely relate to alterations in concentration gradients secondary to changes in cell volume, with consequent ionic movements down their concentration gradients. Luminal hypertonicity is likely to cause cell shrinkage and a consequent increase in intracellular Na+ and Cl–. Because apical Na+ absorption is the predominant ion movement in the basal state, hypertonicity may initially reduce Na+ entry and depolarize the PD. After amiloride, Cl– exit is now favored. Conversely, after the perfusion of a relatively hypotonic solution, cell swelling likely reduces the effective intracellular Cl– concentration and consequently reduces CFTR-mediated Cl– secretion. This suggests a dynamic role for apically located epithelial CFTR. When excessive evaporative losses, such as during exercise, result in a hypertonic, low-volume ASL, a reduction in fluid absorption and increase in fluid secretion are favored. In contrast, an excessive submucosal gland secretion could result in a high-volume hypotonic ASL and, in this situation, further CFTR-mediated Cl– secretion would be inhibited. We conclude that CFTR is involved in the responses to both hypertonicity and hypotonicity. These responses are consistent with a physiologic role for CFTR as an osmoregulator.
Supported by a grant from the Cystic Fibrosis Trust (M.G.D.). Conflict of Interest Statement: M.G.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.M.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.W.F.W.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form October 19, 2003; accepted in final form December 15, 2004
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