The Effect of Varying Tonicity on Nasal Epithelial Ion Transport in Cystic Fibrosis
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美国呼吸和危急护理医学 2005年第4期
Department of Gene Therapy, Faculty of Medicine, Imperial College at the National Heart and Lung Institute, London, United Kingdom
ABSTRACT
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 (5eC7).
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 (10eC12). 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 CleC 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).
METHODS
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
Nasal PD was measured using a previously described method (21). Briefly, the exploring electrode consisted of a size-8 pediatric Foley urinary catheter, which was modified to produce a double-lumen tube. One lumen was filled with electrocardiographic electrode cream diluted with Ringer's saline and a silvereCsilver chloride electrode (SLE Instruments, South Croyden, UK) was placed in the electrode cream in the end of the tube. The second lumen was perfused with the solutions described later at a constant rate of 4 ml/minuteeC1. The reference electrode consisted of a second silvereCsilver chloride electrode placed over an area of abraded skin on the forearm. Electrical offset values up to ± 1 mV were accepted and corrected before obtaining PD values. The exploring electrode was introduced slowly into the nasal cavity and passed along the floor of the nose until the maximum PD was recorded. All subsequent measurements were undertaken at this site.
Solutions
The standard nasal perfusion protocol consisted of a physiologic solution of 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (HEPES), 142 mM CleC, 130 mM Na+, 6 mM K+, 2 mM Ca2+, 1 mM Mg2+, and 10 mM glucose in deionized water (osmolality 301 milliosmoles [mOsm]). After amiloride (100 e) perfusion for 3 minutes, the solution was substituted for a low-chloride solution consisting of the following: amiloride 100 e, 10 mM HEPES, 6 mM CleC, 136 mM gluconate, 130 Na+, 6 mM K+, 2 mM Ca2+, 1 mM Mg2+, and 10 mM glucose in deionized water (osmolarity 301 mOsm). Finally, low chloride plus isoproterenol (100 e) and then ATP (100 e) were perfused.
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 CleC 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 CleC concentrations to assess the magnitude of CleC 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 e, 10 mM HEPES, 40 mM CleC, 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
All drugs were obtained from Sigma Aldrich (Poole, UK), except amiloride, which was a gift from Merck, Sharp, and Dohme (Hoddesdon, UK). Drugs were used at the following final concentrations: amiloride 100 e, isoproterenol 100 e, and ATP 100 e.
Statistical Analysis
Intergroup differences were assessed using the Mann-Whitney U test. Data are expressed as mean ± SEM for convenience. The null hypothesis was rejected at p < 0.05. For discussion purposes, increases and decreases refer to the absolute magnitude of the PD, which was always lumen negative.
RESULTS
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 eC37.8 ± 2.8 mV, non-CF eC17.2 ± 0.76 mV; p < 0.001) and failure of cAMP-mediated CleC secretion (CF eC19.7 ± 1.3 mV, non-CF +1.4 ± 2.1 mV; p < 0.001).
CFTR Is Involved in the Response to Hypertonicity
After perfusion of the standard HEPES solution, the addition of 500 mM mannitol resulted in a similar absolute depolarization in subjects with and without CF (Figure 1). These depolarizations represented 57 (non-CF) and 15% (CF) of the response to amiloride seen in the standard protocols.
After amiloride pretreatment, in the non-CF group, mannitol now produced a hyperpolarization (PD eC 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 CleC were compared with an isotonic solution with the same CleC concentration. In subjects without CF, perfusion of the isotonic 40 mM CleC solution resulted in a relative hyperpolarization (PD eC 6.7 ± 0.7 mV), as expected. In contrast, the hypotonic 40 mM CleC 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 CleC conductance was seen in response to the isotonic 40 mM CleC solution (PD eC 1.2 ± 1.1 mV). The hypotonic 40 mM CleC 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.
DISCUSSION
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 CleC 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 CleC secretion could be measured in subjects without CF, at a CleC concentration commensurate with a hypotonic solution. By keeping the subsequent hypotonic challenge at this constant CleC 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 CleC caused a relative hyperpolarization in the non-CF group but not in the CF group, consistent with the widely reported failure of apical CleC 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 CleC. 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, CleC exit is now favored. Conversely, after the perfusion of a relatively hypotonic solution, cell swelling likely reduces the effective intracellular CleC concentration and consequently reduces CFTR-mediated CleC 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 CleC 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.
REFERENCES
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Knowles MR, Robinson JM, Wood RE, Pue CA, Mentz WM, Wager GC, Gatzy JT, Boucher RC. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J Clin Invest 1997;100:2588eC2595.
Landry JS, Landry C, Cowley EA, Govindaraju K, Eidelman DH. Harvesting airway surface liquid: a comparison of two techniques. Pediatr Pulmonol 2004;37:149eC157.
Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998;95:1005eC1015.
Song Y, Thiagarajah J, Verkman AS. Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways. J Gen Physiol 2003;122:511eC519.
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Wine JJ. The genesis of cystic fibrosis lung disease. J Clin Invest 1999;103:309eC312.
Smith JJ, Travis SM, Greenberg EP, Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 1996;85:229eC236.
Anderson SD, Daviskas E, Smith CM. Exercise-induced asthma: a difference in opinion regarding the stimulus. Allergy Proc 1989;10:215eC226.
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Alsuwaidan S, Li Wan PA, Morrison G, Redmond A, Dodge JA, McElnay J, Stewart E, Stanford CF. Effect of exercise on the nasal transmucosal potential difference in patients with cystic fibrosis and normal subjects. Thorax 1994;49:1249eC1250.
Hebestreit A, Kersting U, Basler B, Jeschke R, Hebestreit H. Exercise inhibits epithelial sodium channels in patients with cystic fibrosis. Am J Respir Crit Care Med 2001;164:443eC446.
Jacquot J, Puchelle E, Hinnrasky J, Fuchey C, Bettinger C, Spilmont C, Bonnet N, Dieterle A, Dreyer D, Pavirani A. Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands. Eur Respir J 1993;6:169eC176.
Egan M, Flotte T, Afione S, Solow R, Zeitlin PL, Carter BJ, Guggino WB. Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR. Nature 1992;358:581eC584.
Gregory RJ, Cheng SH, Rich DP, Marshall J, Paul S, Hehir K, Ostedgaard L, Klinger KW, Welsh MJ, Smith AE. Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 1990;347:382eC386.
Jiang Q, Mak D, Devidas S, Schwiebert EM, Bragin A, Zhang Y, Skach WR, Guggino WB, Foskett JK, Engelhardt JF. Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor. J Cell Biol 1998;143:645eC657.
Willumsen NJ, Davis CW, Boucher RC. Selective response of human airway epithelia to luminal but not serosal solution hypertonicity: possible role for proximal airway epithelia as an osmolality transducer. J Clin Invest 1994;94:779eC787.
Vazquez E, Nobles M, Valverde MA. Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels. Proc Natl Acad Sci USA 2001;98:5329eC5334.
Middleton PG, Geddes DM, Alton EW. Protocols for in vivo measurement of the ion transport defects in cystic fibrosis nasal epithelium. Eur Respir J 1994;7:2050eC2056.
Davies MG, Geddes DM, Alton EW. The effect of varying tonicity on nasal epithelial ion transport in cystic fibrosis. Pediatr Pulmonol 1999;S19:165.
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ABSTRACT
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 (5eC7).
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 (10eC12). 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 CleC 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).
METHODS
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
Nasal PD was measured using a previously described method (21). Briefly, the exploring electrode consisted of a size-8 pediatric Foley urinary catheter, which was modified to produce a double-lumen tube. One lumen was filled with electrocardiographic electrode cream diluted with Ringer's saline and a silvereCsilver chloride electrode (SLE Instruments, South Croyden, UK) was placed in the electrode cream in the end of the tube. The second lumen was perfused with the solutions described later at a constant rate of 4 ml/minuteeC1. The reference electrode consisted of a second silvereCsilver chloride electrode placed over an area of abraded skin on the forearm. Electrical offset values up to ± 1 mV were accepted and corrected before obtaining PD values. The exploring electrode was introduced slowly into the nasal cavity and passed along the floor of the nose until the maximum PD was recorded. All subsequent measurements were undertaken at this site.
Solutions
The standard nasal perfusion protocol consisted of a physiologic solution of 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (HEPES), 142 mM CleC, 130 mM Na+, 6 mM K+, 2 mM Ca2+, 1 mM Mg2+, and 10 mM glucose in deionized water (osmolality 301 milliosmoles [mOsm]). After amiloride (100 e) perfusion for 3 minutes, the solution was substituted for a low-chloride solution consisting of the following: amiloride 100 e, 10 mM HEPES, 6 mM CleC, 136 mM gluconate, 130 Na+, 6 mM K+, 2 mM Ca2+, 1 mM Mg2+, and 10 mM glucose in deionized water (osmolarity 301 mOsm). Finally, low chloride plus isoproterenol (100 e) and then ATP (100 e) were perfused.
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 CleC 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 CleC concentrations to assess the magnitude of CleC 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 e, 10 mM HEPES, 40 mM CleC, 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
All drugs were obtained from Sigma Aldrich (Poole, UK), except amiloride, which was a gift from Merck, Sharp, and Dohme (Hoddesdon, UK). Drugs were used at the following final concentrations: amiloride 100 e, isoproterenol 100 e, and ATP 100 e.
Statistical Analysis
Intergroup differences were assessed using the Mann-Whitney U test. Data are expressed as mean ± SEM for convenience. The null hypothesis was rejected at p < 0.05. For discussion purposes, increases and decreases refer to the absolute magnitude of the PD, which was always lumen negative.
RESULTS
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 eC37.8 ± 2.8 mV, non-CF eC17.2 ± 0.76 mV; p < 0.001) and failure of cAMP-mediated CleC secretion (CF eC19.7 ± 1.3 mV, non-CF +1.4 ± 2.1 mV; p < 0.001).
CFTR Is Involved in the Response to Hypertonicity
After perfusion of the standard HEPES solution, the addition of 500 mM mannitol resulted in a similar absolute depolarization in subjects with and without CF (Figure 1). These depolarizations represented 57 (non-CF) and 15% (CF) of the response to amiloride seen in the standard protocols.
After amiloride pretreatment, in the non-CF group, mannitol now produced a hyperpolarization (PD eC 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 CleC were compared with an isotonic solution with the same CleC concentration. In subjects without CF, perfusion of the isotonic 40 mM CleC solution resulted in a relative hyperpolarization (PD eC 6.7 ± 0.7 mV), as expected. In contrast, the hypotonic 40 mM CleC 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 CleC conductance was seen in response to the isotonic 40 mM CleC solution (PD eC 1.2 ± 1.1 mV). The hypotonic 40 mM CleC 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.
DISCUSSION
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 CleC 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 CleC secretion could be measured in subjects without CF, at a CleC concentration commensurate with a hypotonic solution. By keeping the subsequent hypotonic challenge at this constant CleC 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 CleC caused a relative hyperpolarization in the non-CF group but not in the CF group, consistent with the widely reported failure of apical CleC 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 CleC. 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, CleC exit is now favored. Conversely, after the perfusion of a relatively hypotonic solution, cell swelling likely reduces the effective intracellular CleC concentration and consequently reduces CFTR-mediated CleC 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 CleC 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.
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