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Airway Surface Liquid Calcium Modulates Chloride Permeability in the Cystic Fibrosis Airway

Cystic Fibrosis Unit, Department of Respiratory Medicine, Westmead Hospital, Westmead, New South Wales, Australia; and Department 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 Peter G. Middleton, M.B.B.S., B.Sc. (Med), Ph.D., F.R.A.C.P., University of Sydney, Cystic Fibrosis Unit, Department of Respiratory Medicine, Westmead Hospital, Westmead 2145, New South Wales, Australia. E-mail: peterm{at}westgate.wh.usyd.edu.au

	ABSTRACT

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Patients with cystic fibrosis (CF) demonstrate a characteristic defect in epithelial chloride movement, which can be demonstrated in vivo by the nasal potential difference technique. After amiloride pretreatment, the CF airway exhibits only a transient response to perfusion with low-chloride solution, contrasting with the sustained hyperpolarization seen in control subjects. This study further investigated the response to low-chloride solution in the CF airway, examining the interaction between surface divalent ions and the low-chloride response. Sequential perfusion with amiloride, low chloride, and isoproterenol was tested in groups of subjects with CF, with the diluent containing different concentrations of calcium and magnesium, on different days. When the low-chloride response was measured with the nominally calcium-free diluents, the subjects with CF had mean (SEM) responses of 8.0 (0.7), 8.6 (2.4), and 9.6 (1.6) mV in the presence of 0, 1, and 3 mM Mg²⁺, respectively, significantly different from the response in the presence of divalent ions. However, the subsequent response to isoproterenol was not different in the presence or absence of divalent ions. We hypothesize that perfusion of the CF airway with nominally calcium-free solutions reduces tonic inhibition of chloride secretion.

Key Words: cystic fibrosis • potential difference • chloride • calcium

The clinical syndrome of cystic fibrosis (CF) comprises recurrent sinobronchial infections, pancreatic fibrosis, bowel dysfunction, and increased sweat electrolytes. Chronic suppurative lung disease leading to respiratory failure is the major cause of morbidity and mortality. After cloning of the CF transmembrane conductance regulator gene (CFTR), numerous studies have shown that CFTR functions both as a cyclic AMP–regulated Cl^- channel (1) and as a regulator of other ion channels (2).

The airway epithelium of subjects with CF demonstrates decreased Cl^- and increased Na⁺ transport, thought to be involved in disease pathogenesis. As the primary genetic defect in CF relates to Cl^- movement, numerous studies have attempted to induce Cl^- movement as a potential new treatment for the disease. However, CFTR is not the sole Cl^- channel present in airway epithelial cells. Other Cl^- channels regulated by intracellular Ca²⁺ or changes in cell volume appear to function normally in CF cells when assessed in vitro (3). These "alternate" Cl^- channels may therefore provide a mechanism to bypass the Cl^- transport defect in CF. Preliminary trials have already demonstrated that topical application of adenosine triphosphate (ATP) can induce Cl^- secretion in human subjects in vivo (4).

As the respiratory epithelium lining the human nose, trachea, and lower airways shows similar ion transport processes, the nose is widely used as a model for the human lower airways. We, and others, have demonstrated previously that the nasal potential difference (PD) technique reliably measures both abnormalities of ion transport characteristically found in CF (5, 6). The increased Na⁺ absorption is reflected in an increase in resting unstimulated PD, together with an increased response to the Na⁺ channel blocker amiloride (7). The defect in Cl^- movement across the CF airway is evident only after stimulation of Cl^- secretion (6). Perfusion with a low-Cl^- solution, in the presence of amiloride, induces a rapid and sustained response in subjects without CF, and subsequent perfusion with the cyclic AMP–elevating agent isoproterenol further increases PD. In contrast, the CF airway demonstrates only small transient responses, with no sustained component measured at 5 minutes. Thus, the nasal PD responses to low-Cl^- and isoproterenol solutions provide a convenient surrogate marker of the basic CF defect. However, it is not apparent why the CF airway does not secrete Cl^- through the alternate Cl^- channels in response to the nonspecific stimulus of a low-Cl^- solution.

In a pilot study, we noted that perfusion with divalent-free amiloride/low-Cl^-/isoproterenol solution induced a marked nasal PD response in 2 subjects with CF (data not shown). In the current study, we have investigated the effect of manipulation of the Ca²⁺ and Mg²⁺ concentrations on the response to low-Cl^- and isoproterenol solutions and have demonstrated that the CF airway can respond to a low-Cl^- stimulus when the airway surface liquid is (nominally) calcium-free. Some of these results have been reported previously as abstracts (8, 9).

	METHODS

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Subjects
Subjects with CF (aged 17–32 years) with typical clinical syndromes and abnormal sweat electrolytes were recruited. All were pancreatic insufficient and were tested during a period of clinical stability at least 4 weeks after an upper respiratory tract infection. The study was approved by the Hospital Ethics Committee, and all subjects gave written informed consent.

Nasal PD Measurements
Nasal PD was measured using methods described previously (6, 7, 10). Briefly, the exploring electrode consisted of a double lumen silicone rubber tube with the openings of both lumens at the same site, 3 mm from the tip. One lumen was filled with an equal mixture of saline and ECG electrode cream, connected to a high impedance voltmeter via a silver–silver chloride electrode. The second lumen was perfused with the different solutions as outlined subsequently, using a peristaltic pump that provided a continuous flow of 4 ml/minute throughout the perfusion period. The reference electrode consisted of a second silver–silver chloride electrode placed over an area of abraded skin on the forearm, again connected to the voltmeter. Before recordings, the offset of the electrodes was measured and appropriate corrections made to recorded values.

Perfusion commenced with Krebs solution of composition (mM): Na⁺ 140, K⁺ 6, Ca²⁺ 2, Mg²⁺ 1, Cl^- 152, glucose 10, and N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid 10, titrated to pH 7.4, designated in this study as Krebs (2:1) to indicate Krebs containing 2 mM Ca²⁺ and 1 mM Mg²⁺. Low-Cl^- solutions (6 mM) were prepared by substituting NaCl and KCl with equimolar gluconate; when required, CaCl₂ and MgCl₂ were replaced with NaCl to maintain Cl^- concentrations. All solutions were perfused sequentially, and due to the 3-ml dead space, the new perfusate reached the catheter tip approximately 45 seconds after solution change. Fresh stock solutions were prepared daily and diluted as required. All solutions were perfused at room temperature (21–23°C). The term "divalent-free" is used for the nominally divalent-free solutions where no Ca²⁺ or Mg²⁺ has been added.

The "standard" perfusion protocol comprised successive addition of amiloride (100 µM), low-Cl^- solution, and finally isoproterenol (10 µM), each for a period of 5 minutes as described previously (6). On different days, the standard protocol was repeated with the diluent containing different divalent ion concentrations. In all cases, the tests started with an initial period of stabilization using standard Krebs (2:1) solution. After stabilization of the nasal PD, the diluent was changed to Krebs N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid with divalent concentrations (Ca²⁺:Mg²⁺) of either (0:0), (2:0), (0:1), or (0:3) and the protocol repeated. Different divalent ion responses were performed on different days in random order.

Statistical Analysis
Data are expressed as mean (SEM) and the unpaired two-tailed Student's t test was used for comparison; 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

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Effect of Divalent-free Solutions on Epithelial Integrity and Sodium Transport
When the normal Krebs (2:1) was changed to divalent-free solution (0:0), the mean (SEM) nasal PD in 7 subjects with CF gradually increased (became more negative) over the next 5 minutes by 3.5 (1.2) mV. This was not statistically different from a "sham" change of diluent, i.e., from Krebs (2:1) to (2:1), perfused for a further 5 minutes, which increased PD by 2.7 (2.1) mV. The amiloride response in the absence of divalent ions Krebs (0:0) was -23.5 (3.1) mV, which was not statistically different from that in the presence of divalent ions -30.5 (3.6) mV, p = not significant (Table 1) . Together, these results suggest that perfusion with divalent-free solution neither compromises the integrity of the airway epithelium nor significantly alters amiloride-sensitive Na⁺ transport.

View larger version (26K): [in this window] [in a new window]   Figure 1. Mean (SEM) responses to perfusion with low-Cl- (6 mM) and isoproterenol (10 µM) in the presence, Krebs (2:1), or in the absence, Krebs (0:0), of divalent ions in subjects with CF, tested on different days (n = 8, n = 7, respectively). For comparison, the response in 20 subjects without CF in the presence of divalent ions is also shown. Low-Cl- solution was commenced at time 0 and isoproterenol at time 5 minutes, but because of the dead space in the system, the solutions only reached the nasal epithelium after approximately 45 seconds. Amiloride (100 µM) was commenced 5 minutes before the low-Cl- solution and continued throughout the recordings.

The responses to low Cl^- and isoproterenol in subjects with CF for the different solutions were next assessed. Removal of Ca²⁺ alone (0:1) mimicked the response to divalent free solution (0:0). Removal of magnesium alone (2:0) was associated with a small but statistically significant increase in the low-Cl^- response to 4.2 (1.8) mV (p < 0.05 vs. divalent containing solution). To delineate the relative effects of Ca²⁺ and Mg²⁺, we then measured the low-Cl^- response in Krebs (0:3) solution. Responses were indistinguishable from either of the other Ca²⁺-free solutions (0:0) or (0:1) (Figure 2) , suggesting that lack of Ca²⁺ is predominantly responsible for the low-Cl^- responses in subjects with CF.

View larger version (18K): [in this window] [in a new window]   Figure 2. Mean (SEM) responses to perfusion with low-Cl- (6 mM) and isoproterenol (10 µM) in the presence of different concentrations of divalent ions, as shown. Low-Cl- solution was commenced at time 0, and isoproterenol at time 5 minutes, but because of the dead space in the system, the solutions only reached the nasal epithelium after approximately 45 seconds. Amiloride (100 µM) was commenced 5 minutes before the low-Cl- solution and continued throughout the recordings. (A) Responses to (2:1) filled squares, (0:1) open circles, (0:3) open triangles. (B) Responses to (0:1) filled diamonds, (2:0) filled triangles.

	DISCUSSION

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This study has demonstrated that, if the airway surface liquid is nominally divalent-free, the CF airway epithelium can respond to a low-Cl^- solution with a sustained hyperpolarization of 8 to 10 mV. This compared with responses in the presence of divalent ions of 0–1 mV in subjects with CF and with previously published responses of 15 to 20 mV in subjects without CF (6). The similar responses found in subjects with CF when the perfusate contained 0 mM Ca²⁺ and either 0, 1, or 3 mM Mg²⁺ suggests that the Ca²⁺ concentration in the ASL is the most important variable. To our knowledge, this is the first demonstration that apical (extracellular) divalent ions can modulate the response to low-Cl^- solution in the CF airway.

There are a number of possible effects that could be induced by the removal of Ca²⁺ ions from the ASL. Numerous in vitro studies have demonstrated that perfusion with nominally Ca²⁺-free solutions can alter the integrity of apical tight junctions. Marin and Zaremba (11) measured the effect of Ca²⁺-free solutions on ion transport across canine tracheal epithelium mounted in Ussing chambers. Removal of Ca²⁺ from both sides of the epithelium decreased PD, predominantly via a decrease in epithelial resistance; the effect of apical versus basolateral Ca²⁺-free solutions was not reported. In contrast, the current study found no evidence of such an effect as the resting (unstimulated) PD remained stable or slightly increased after perfusion with Ca²⁺-free Krebs. As disruption of the tight junctions would have reduced the epithelial resistance, and hence the PD, it is unlikely that perfusion of the human airway with nominally Ca²⁺-free solution for the time period studied resulted in epithelial damage.

Another potential effect of the Ca²⁺-free solution may be alteration of Na⁺ absorption. Curran and Gill (12) demonstrated that addition of Ca²⁺ to the fluid bathing the outer (apical) surface of the frog skin decreased short-circuit current, through decreased apical Na⁺ absorption. In that model, addition of Mg²⁺ also decreased Na⁺ absorption, though the response was smaller than that associated with Ca²⁺. More recently, other investigators have demonstrated that intracellular but not extracellular Ca²⁺ can modulate Na⁺ absorption. Amiloride-insensitive, calcium-dependent nonselective cation channels were demonstrated in human nasal epithelial cells in vitro, although these channels were found only in approximately 4% of cell-attached patches (13). However, in the current study, the stable resting PD and the lack of a consistent change in amiloride response after perfusion with Ca²⁺-free solution suggest that removal of extracellular Ca²⁺ does not significantly modulate the Na⁺ channels present in the apical membrane of the human airway epithelium in vivo.

In contrast to the lack of change in the measurements of Na⁺ absorption, the current study demonstrated a significant increase in the Cl^- secretory response when the apical surface of the CF airway was made nominally Ca²⁺-free. This suggests that the Ca²⁺ ions in the ASL may be involved in modulation of Cl^- movement in the CF airway. As the current study was performed in human subjects in vivo, we were not able to use specific Cl^- channel blockers to delineate the channel(s) involved. However, the lack of a subsequent isoproterenol response suggests that the cyclic AMP–regulated Cl^- channels, such as CFTR and the outwardly rectifying Cl^- channel, are not involved. We hypothesize that removal of Ca²⁺ ions allows one or more of the alternate Cl^- channels to respond to the nonspecific stimulus of perfusion with low-Cl^- solution. This suggests that the channel is tonically inhibited by apical surface Ca²⁺ and that Ca²⁺-free solutions remove this inhibition, allowing the CF airway to respond to low-Cl^- solution.

Recently it was demonstrated that removal of extracellular Ca²⁺ could activate a Cl^- current in chicken ovarian granulosa cells (14). Using whole-cell patch clamp recordings, it was shown that removal of Ca²⁺ from the bath was associated with an increase in a transient outward current. This current was reduced by Cl^- channel blockers but was not altered by changes in intracellular Ca²⁺. A Ca²⁺-inactivated Cl^- channel has also been described in Xenopus oocytes (15), although the biophysical properties were slightly different from those described in the ovarian granulosa cells. Removal of extracellular Ca²⁺ may decrease intracellular Ca²⁺; increased intracellular Ca²⁺ has been shown to decrease the open probability of cyclic nucleotide–gated ion channels in olfactory neurons (16), although whether the converse also occurs is not known. As the current study demonstrated that the response to isoproterenol was unchanged by the different divalent solutions, it would appear more likely that the response to low-Cl^- solution is mediated by the alternate Cl^- channels. Finally, the Ca²⁺-free solution may alter ion transport via intermediate steps, such as changes in extracellular nucleotidase activity, which, by altering ATP levels, may then change Cl^- responses through the alternate Cl^- channels.

We hypothesize that the endogenous Ca²⁺ present in the ASL blocks the alternate Cl^- channels from responding to nonspecific stimuli for Cl^- secretion. This may explain the apparent inability of the alternate Cl^- channels to mediate Cl^- secretion in subjects with CF, despite the evidence that these channels are expressed and function normally in CF cells in vitro. As the Ca²⁺ concentration of the ASL of the human trachea has previously been measured in the range of 1 to 4 mM both in subjects with CF and without CF (17, 18), this endogenous level of Ca²⁺ in the ASL would appear sufficient to block the alternate Cl^- channels from responding to a nonspecific Cl^- stimulus.

In conclusion, we have demonstrated that perfusion with nominally Ca²⁺-free solutions alters the response to low-Cl^- solution in subjects with CF. This suggests that airway surface calcium exerts tonic inhibition on Cl^- secretion in the human airway.

	Acknowledgments

 
The authors thank the subjects who took part in this study.

	FOOTNOTES

 
Supported by the Australian Cystic Fibrosis Research Trust, New South Wales Government Employees Medical Research Fund, Asthma Foundation of New South Wales, and by an NH&MRC Postgraduate Medical Research Fellowship (P.G.M.) and a Wellcome Trust Senior Clinical Fellowship (E.W.A.).

Conflict of Interest Statement: P.G.M. has no declared conflict of interest; K.A.P. has no declared conflict of interest; E.D. has no declared conflict of interest; J.R.W. has no declared conflict of interest; D.M.G. has no declared conflict of interest; E.W.A. has no declared conflict of interest.

Received in original form May 9, 2003; accepted in final form August 20, 2003

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