Basal chloride currents in murine airway epithelial cells: modulation by CFTR

R. Tarran1, M. A. Gray1, M. J. Evans2, W. H. Colledge2, R. Ratcliff2, and B. E. Argent1

1 Department of Physiological Sciences, University Medical School, Newcastle upon Tyne NE2 4HH; and 2 Wellcome/Cancer Research Campaign Institute of Cancer and Developmental Biology, Cambridge CB2 1QR, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl- conductances and, under certain conditions, a small Na+ conductance. Voltage-clamp experiments revealed three distinct Cl- conductances. Itv-indep was time and voltage independent with a linear current-voltage (I-V) plot; Iv-act exhibited activation at potentials greater than ±50 mV, giving an S-shaped I-V plot; and Ihyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I-V plot. The current density sequence was Ihyp-act = Iv-act >>  Itv-indep. These conductances had Cl--to-N-methyl-D-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. Itv-indep and Iv-act were present in wild-type and CF cells at equal density and frequency. However, Ihyp-act was detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).

nasal epithelial cells; chloride conductance; patch-clamp technique; cystic fibrosis; transgenic mice; cystic fibrosis transmembrane conductance regulator

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE PROXIMAL AIRWAYS of the lung are lined with a layer of fluid that serves to trap airborne particles and bacteria. The composition and thickness of this fluid layer are regulated by the rates of Na+ absorption and Cl- secretion across the airway epithelium and are critical for effective mucocilliary clearance and bacterial killing (21). In the inherited disease cystic fibrosis (CF), the airways lack cAMP-stimulated Cl- secretion (1) due to the absence, or defective functioning, of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. However, CF airways also exhibit an increased apical Na+ conductance (1), which is explained by an upregulation of amiloride-sensitive Na+ channels in response to the absence of CFTR (1, 22). Combined, these alterations in Cl- and Na+ conductances are thought to result in desiccation of the airways, which leads to bacterial colonization and inflammatory damage and, ultimately, lung failure.

The airway epithelium contains at least three stimulus-activated Cl- conductance pathways: 1) the CFTR conductance that is activated by cAMP, 2) a Ca2+-activated conductance, and 3) a volume-activated conductance (24). The biophysical properties of these stimulus-activated conductances are quite distinct and are similar in both airway cells (24) and other epithelia that are affected by CF (6, 28). In addition to these stimulus-activated conductances, there is also evidence for the existence of a basal (i.e., unstimulated) Cl- conductance in the airway epithelium (3, 10, 14, 25, 26). This basal Cl- conductance may act as a transcellular route for the Cl- flux that must accompany Na+ absorption across the airway epithelium.

Chan et al. (3) reported that the basal Cl- conductance in cultured human airway cells was due to a time-dependent current that had either a linear or outwardly rectified current-voltage (I-V) in different cells. However, a detailed characterization of the biophysical and pharmacological properties of the basal Cl- conductance in respiratory cells has not been performed. Therefore, this was the main aim of the present work. In particular, we sought to answer the following questions: 1) is the basal Cl- conductance a distinct entity or does it simply represent the background activity of one or more of the stimulus-activated conductances already known to exist in the airway epithelium, and 2) is the basal Cl- conductance affected by the absence of CFTR? To address these questions, we have used ciliated respiratory cells because they are thought to play an important role in proximal airway ion transport (1) and have been shown to express CFTR (2). We decided not to use cultured cells but instead have developed a procedure for the isolation of ciliated respiratory cells, which are suitable for patch clamping, from the murine nasal epithelium (NE). In this study, we demonstrate that, in the absence of any agonists, one of three distinct spontaneously active Cl- conductances may be active in these ciliated cells. In addition, our data provide evidence suggesting that one of these basal Cl- conductances is modulated by CFTR.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

Mice of either sex were used for the experiments. Transgenic CF null mice (cftrtm1cam) with a disrupted cftr gene were created by targeted replacement mutagenesis as previously described (17). In brief, the replacement plasmid vector pCF-B(hprt)tk was used to disrupt the CF gene in exon 10, which encodes part of the first nucleotide binding fold (NBF) of CFTR (17). The hprt minigene introduces a termination codon that prematurely terminates the CFTR protein within the first NBF. Wild-type (WT) mice were obtained either from the Cambridge transgenic breeding colony or from a BALB/c breeding colony at the University of Newcastle upon Tyne. Because identical results were obtained from both groups of WT animals, these data have been combined. A total of 15 WT [4 Cambridge (2 males and 2 females) and 11 BALB/c (6 males and 5 females)] and 6 CF null animals (5 males and 1 female) were used in this study. The average age and weight of the animals were 63 ± 7 days old and 25.0 ± 0.7 g (for WT) and 53 ± 4 days old and 17.5 ± 2.3 g (for cftrtm1cam).

Isolation of Ciliated Respiratory Cells From the NE

Ciliated respiratory cells, suitable for patch clamping, were obtained from the NE using a two-stage isolation technique. First, the NE was removed from the mouse using a modification of the method described by Grubb et al. (10). Then, after an overnight incubation in protease, single cells and small groups of cells were teased from the NE. In the mouse, the nasal membrane contains four types of epithelia: respiratory, olfactory, transitional, and squamous (15). Most of the respiratory epithelium is located in the lateral and ventral regions of the nasal cavity (15).

Mice were killed by cervical dislocation, and the NE was removed immediately. Throughout the dissection, the preparation was viewed at ×7 magnification using a dissecting microscope. First, the skin over the nose was removed with scissors, and the tissue covering the nasal bones was cut away with a scalpel. The nasal bones were then gripped with forceps at the end closest to the tip of the nose and lifted away, revealing the two pieces of NE on either side of the nasal septum. To remove any blood and debris, the nasal cavity was washed with DMEM containing 1% penicillin-streptomycin (stock contained 103 units penicillin and 10 mg streptomycin/ml; Sigma), a procedure that also lifted the NE away from the nasal cavity, making it easier to remove. Each piece of NE was then dissected from the nasal cavity using forceps and scissors. The NE was first cut at the end closest to the brain and carefully lifted away using forceps. If the NE still appeared to be attached to the septum laterally [where most of the respiratory epithelium is located (15)], it was carefully dissected away with either scissors or a scalpel. Finally, the NE was cut at the end closest to the tip of the nose and removed. The isolated NE was washed in DMEM containing antibiotics and then placed in an Eppendorf tube containing 1 ml of the same solution plus 0.05% wt/vol protease XIV (Sigma) and incubated for 24 h at 4°C. The enzyme activity was stopped by spinning the Eppendorf tube at 14 g for 30 s, removing the supernatant, and resuspending the tissue in DMEM containing antibiotics plus 10% fetal calf serum at 4°C.

To isolate ciliated respiratory cells suitable for patch clamping, the protease-treated NE was viewed under phase-contrast optics (×200), and areas of the tissue that contained cells with beating cilia were identified. Small pieces of NE, containing 50-100 beating cells, were then separated from the main body of the tissue using sharpened stainless steel needles and transferred, using a micropipette, to a microwell containing ~150 µl of DMEM. The microwell was then transferred to a dissecting microscope (×45 magnification), and the small pieces of NE were teased apart using sharpened entomology pins to yield single cells and small groups of cells. The respiratory epithelial cells could easily be distinguished from other cell types by their prominent cilia (Fig. 1). Our criteria for cell viability were as follows: 1) a clear, bright, phase-contrast image and 2) beating cilia. On the basis of these criteria, the majority of isolated respiratory cells were viable.


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Fig. 1.   Scanning electron micrograph image of a respiratory cell isolated from the mouse nasal epithelium. Note the prominent cilia on the apical membrane. Scale bar = 2 µm.

To check whether incubating the NE in protease for 24 h at 4°C affected the electrophysiological properties of the isolated cells, we also studied cells isolated from NE that had been exposed to the enzyme for only 1 h. Both groups of cells had similar electrophysiological characteristics, suggesting that the 24-h incubation does not have a detrimental effect. Moreover, the yield of viable cells was much lower when a 1-h protease incubation was employed, so most of our electrophysiological data come from cells isolated using the longer incubation time.

The isolated cells remained viable and suitable for electrophysiological experiments for at least 2 h when bathed in the standard Na+-rich bath solution. However, when a bath solution containing N-methyl-D-glucamine (NMDG) chloride was employed, the cells had to be used within ~30 min. Therefore, under these latter conditions, it was necessary to repeat the last stage of the isolation procedure several times during the day to maintain adequate supplies of fresh cells.

Electrophysiology

The cell preparations were transferred to a tissue bath (volume of 1.5 ml) mounted on a Nikon Diaphot inverted microscope and viewed using phase-contrast optics. Because isolated respiratory cells retain their morphology (Fig. 1), have beating cilia, and are often motile, they could easily be distinguished from other cell types. Pipettes were pulled from borosilicate glass (Clarke Electromedical) and had resistances, after fire polishing, of between 2 and 4 MOmega . Gigaohm seals (typically 10-30 GOmega ) were obtained on the nonciliated basolateral membrane of the isolated cells (Fig. 1), with a success rate of ~70% for both WT and CF cells, provided the cells were used within 30 min of their isolation. Formation of seals usually required application of slight suction to the recording pipette. It was not possible to obtain seals on the ciliated apical membrane. Because only the basolateral membrane could be patched and because most of the cells were motile, it was often necessary to apply positive pressure to the patch pipette to orient the cells before attempting to obtain a seal. Membrane potential (Vm) and current recordings were made at room temperature from either single cells or small groups of cells (<= 7), using the whole cell configuration of the patch-clamp technique. We noticed that the cilia beat frequency usually increased when a cell-attached seal was obtained. However, once a whole cell recording was established, the cilia invariably stopped beating, probably because of the low free Ca2+ concentration (<1 nM) in the pipette solution.

Vm (current-clamp experiments) and whole cell currents (voltage-clamp experiments) were recorded with an EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany). To obtain I-V relationships, the Vm was held at 0 mV and then voltage clamped over the range ±100 mV in steps of 20 mV. Each voltage step lasted 500 ms, and there was an 800-ms interval at the holding potential between steps. Data were filtered at 1 kHz and sampled at 2 kHz with a Cambridge electronic design 1401 interface (CED, Cambridge, UK) and stored on the computer hard disk. I-V plots for the time-independent current (Itv-indep) were constructed using the average current measured over a 200-ms period starting 150 ms into the voltage pulse. I-V plots for the two other currents we identified (Iv-act and Ihyp-act) were constructed using the average current measured over a 2-ms period starting 495 ms into the voltage pulse. The currents were not leak corrected. Series resistance (Rs) was typically two to three times the pipette resistance, and Rs compensation (40-50%) was routinely used. Vm have been corrected for current flow across the uncompensated fraction of Rs using the relationship Vm = Vp - IRs, where Vp is the pipette potential and I is current flow. Reversal potentials (Erev) were obtained from I-V plots by interpolation after fitting a third- or fourth-order polynomial using least squares regression analysis. The input capacitance (Ci) of the cells was routinely measured using the analog circuitry of the EPC-7 amplifier and compensated before the recording was started. Ci values were used to calculate current density (which is expressed as pA/pF). Junction potentials were measured, and the appropriate corrections were applied to Vm (27).

Solutions and Chemicals

For measurement of Vm under current-clamp conditions, the pipette solution contained (in mM) 120 KCl, 2.0 EGTA, 2.0 MgCl2, 1.0 ATP, and 10.0 HEPES, pH 7.2 (calculated free Ca2+ concentration of <1 nM). The standard bath solution contained (in mM) 145 NaCl, 4.5 KCl, 2.0 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. The high K+ and sodium aspartate bath solutions contained 145 mM of either KCl or sodium aspartate instead of NaCl.

To isolate anion-selective currents in the voltage-clamp experiments, cation conductances were blocked with NMDG. To prevent the development of swelling-induced Cl- currents, the pipette solution was made 45 mosM hypotonic to the bath solution. The pipette solution contained (in mM) 120 NMDGCl, 2.0 MgCl2, 2.0 EGTA, 1.0 ATP, and 10.0 HEPES, pH 7.2 (calculated free Ca2+ concentration of <1 nM). The standard bath solution contained (in mM) 149.5 NMDGCl, 2.0 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES, pH 7.4. Cl- selectivity of the currents was assessed by Erev shifts following replacement of extracellular Cl-. In the Cl--replacement bath solution, 100 mM NMDGCl was replaced by an isosmotic amount of mannitol. The osmolarities of all solutions were checked using a freezing-point depression osmometer (Roebling, model 10B), and all pipette solutions were filtered (10 µm pore size) before use.

Tamoxifen (10 µM), flufenamic acid (50 µM), glibenclamide (100 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 µM) were made up daily as stock solutions in dimethyl sulfoxide and then diluted 1,000-fold to give the final concentrations employed. DIDS (0.5 mM), tetraethylammonium (TEA; 10 mM), ZnCl2 (0.5 mM), and GdCl3 (0.5 mM) were dissolved directly in the standard bath solution.

Statistics

Significance of difference between means was determined using either analysis of variance followed by Dunn's multiple comparison test (ANOVA + DMCT), the Mann-Whitney U test (M-WUT, nonparametric equivalent of unpaired t-test), or the Wilcoxon's signed rank test (WSRT, nonparametric equivalent of paired t-test) as appropriate. Significance of difference between the number of cells responding to a particular maneuver was assessed using the chi 2 test. The level of significance was set at P <=  0.05. All values are expressed as means ± SE, where n denotes the number of cells used in an experiment. All statistical tests were performed using the numbers of cells rather than the numbers of animals.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Electrical Coupling Between Respiratory Cells

About 50% of the whole cell recordings made in this study were obtained from single ciliated respiratory cells. The Ci values for single WT and CF cells were 17.3 ± 1.3 pF (n = 20 cells from 15 mice) and 18.4 ± 1.5 pF (n = 21 cells from 6 mice), respectively, values that are not statistically different (P = 0.70; M-WUT) (Table 1). The remainder of the experiments were performed on respiratory cells joined together in groups, with up to a maximum of seven cells per group. Table 1 shows that, for both genotypes, cells in groups were not electrically coupled to their neighbors, since there was no correlation between the Ci and the number of cells in a group (P = 0.35; ANOVA). For this reason, electrophysiological data obtained from single isolated cells and from cells in groups have been combined.

                              
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Table 1.   Input capacitance of ciliated nasal epithelial cells from the mouse

Current-Clamp Experiments

The mean resting Vm of the isolated respiratory epithelial cells was 32.2 ± 3.8 mV (n = 22 cells from 3 mice). Figure 2A shows a plot of whole cell conductance (G) against Vm for 22 cells. These data suggest that there might be two populations of respiratory cells in the NE: a group that has a high Vm and a low G (type 1 cells, n = 13/22 cells from 3 mice, 59.1%) and a group that has a low Vm and a high G (type 2 cells, n = 9/22 cells from 3 mice, 40.9%). Type 1 cells had an average Vm and inward G of -43.5 ± 2.7 mV and 0.8 ± 0.03 nS (n = 13), respectively, whereas the values for type 2 cells were -12.5 ± 2.2 mV and 3.11 ± 0.55 nS (n = 9), respectively. The data in Fig. 2A were obtained immediately after establishing a whole cell recording. However, provided the recording could be maintained, Vm values remained stable for at least 8 min and it was still possible to clearly distinguish two populations of cells on the basis of their Vm values at this time point (Fig. 2B). Figure 2C shows basal whole cell currents, measured at the Erev ±60 mV, for the two types of cells. Note that the currents for the type 1 cells are smaller and exhibit marked outward rectification when compared with those measured in type 2 cells (Fig. 2C).


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Fig. 2.   Electrophysiological evidence for the existence of 2 populations of nasal respiratory epithelial cells. These experiments were performed using a K+-rich pipette solution and an Na+-rich bath solution (see METHODS). A: plot of membrane potential (Vm) against whole cell conductance (G). G was calculated as the slope conductance between 0 and -100 mV. Each point represents a separate cell. Two populations of cells are evident: type 1 cells have high Vm and low G; type 2 cells have low Vm and high G. B: Vm at the start of recording and at 2 subsequent time points for type 1 (left) and type 2 (right) cells. C: current densities in the 2 cell groups measured at reversal potential ±60 mV. Open bars, type 1 cells; hatched bars, type 2 cells. Data in A, B, and C are from a total of 22 cells isolated from 3 wild-type (WT) mice.

Initially, we thought that the low Vm and high G values of the type 2 cells might simply be explained by a leaky plasma membrane caused by damage during the isolation procedure. To check for this, we tested the effect of changes in extracellular K+ and Cl- concentrations, and of various ion channel blockers, on the Vm of the isolated cells. Figure 3A shows data for type 1 cells. Note that both increasing extracellular K+ concentration ([K+]o) from 4.5 to 149.5 mM and exposing the cells to 10 mM TEACl (a K+ channel blocker) caused a marked depolarization of Vm. Replacing most of the extracellular Cl- with aspartate also caused a small but statistically significant depolarization, whereas the metal ions Gd3+ and Zn2+ had no effect. Amiloride (100 µM) alone also had no effect on Vm in type 1 cells (Fig. 3A); however, if amiloride was tested after exposure to TEACl and Zn2+, then we observed small hyperpolarizations in three out of four cells (individual values were -8, 0, -1.5, and -1.5 mV). Together, these data suggest that a K+ conductance dominates the membrane of type 1 cells but that the cells also have a small Na+ conductance. The results of similar experiments performed on type 2 cells are shown in Fig. 3B. High [K+]o, TEACl, and replacement of extracellular Cl- with aspartate all caused a significant depolarization of Vm. However, in contrast to type 1 cells, the Vm of type 2 cells was markedly hyperpolarized by Gd3+ and Zn2+. Again, amiloride alone had no effect on Vm (Fig. 3B) but did cause a small hyperpolarization in three out of four type 2 cells that had been previously exposed to TEACl and Zn2+ (individual values were -6, -2, 0, and -0.5 mV). These data suggest that the membrane of type 2 cells possesses both K+ and Cl- conductances plus a small Na+ conductance. The presence of a substantial Cl- conductance most likely explains why type 2 cells have a relatively low resting Vm.


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Fig. 3.   Effect of changes in extracellular K+ and Cl- concentrations and various channel inhibitors on the Vm of nasal respiratory epithelial cells. A: type 1 cells (high Vm and low G). B: type 2 cells (low Vm and high G). Open bars, control; hatched bars, test; filled bars, washout. Number of observations and P values (control vs. test, Wilcoxon's signed rank test) are shown below each data set (ns denotes not significant, P > 0.05). Data were obtained from WT cells. TEACl, tetraethylammonium chloride.

Voltage-Clamp Experiments

Spontaneously active whole cell anion currents. The current-clamp experiments suggested that a proportion of the isolated respiratory cells (the type 2 cells that make up 40.9% of the total) possesses a resting Cl- conductance. We sought to establish the biophysical characteristics of this conductance by measuring whole cell anion currents in voltage-clamp experiments.

Immediately after a whole cell recording was established, one of three different anion-selective currents was detected in WT and CF cells. These currents could be distinguished easily on the basis of their biophysical characteristics. Figure 4A1 shows an example of one of these currents that exhibited no time or voltage dependence over the potential range of ±100 mV, which we have called Itv-indep. Figure 4A2 shows that the steady-state I-V plot for Itv-indep is linear when the standard bath and pipette solutions are employed. A second type of current that we observed was characterized by activation at both positive and negative holding potentials >50 mV (Iv-act). The I-V curve for Iv-act, measured at the end of the voltage step, was sigmoidal (Fig. 4B2). Figure 4C1 shows the third type of current, which displayed slight inactivation at positive potentials and marked activation at negative potentials (Ihyp-act). The I-V plot for this current was inwardly rectified with the standard pipette and bath solutions (Fig. 4C2). There were no obvious morphological differences between cells that exhibited the different current types. Indeed, single cells that had been isolated from the same cell clump, and which appeared identical, often expressed different currents.


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Fig. 4.   Biophysical characteristics of spontaneously active Cl- currents in nasal respiratory epithelial cells. A1-C1: typical whole cell currents recorded by holding the Vm at 0 mV and pulsing between ±100 mV in 20-mV steps. A2-C2: current-voltage plots obtained using this voltage protocol. black-square, Standard conditions; bullet , replacement of 100 mM bath N-methyl-D-glucamine chloride with mannitol. A: time- and voltage-independent current (Itv-indep). B: current that activates at both positive and negative potentials (Iv-act). C: hyperpolarization-activated current (Ihyp-act). Data were obtained from WT cells.

All three conductances were found to reverse close to the predicted equilibrium potential for Cl- (-5.7 mV), and Cl- selectivity was confirmed by the positive shift in Erev following replacement of 100 mM NMDGCl in the bath solution with mannitol (Table 2). The conductances were between 2.8 and 10.3 times more permeable to Cl- than to NMDG+ (Table 2), with Itv-indep and Ihyp-act having the highest Cl- selectivity and Iv-act having the lowest (Table 2). There were no differences in the calculated Cl--to-cation permeability ratios for the currents observed in WT and CF cells (Table 2).

                              
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Table 2.   Cl- selectivity of spontaneously active whole cell currents in mouse ciliated nasal epithelial cells

Finally, some cells exhibited a small current on break-in (<50 pA), which reversed at -0.9 ± 10.7 mV (n = 11 cells from 6 mice) and which was not Cl- selective as judged by the lack of shift in Erev after bath Cl- substitution. We called this small, non-anion-selective current Ins.

Densities of spontaneously active currents in WT and CF cells. Figure 5 compares the densities of the various spontaneously active currents, measured on average 44 ± 3 s after a whole cell recording was established, in WT and CF nasal cells. The current density sequence for both WT and CF cells was the same, Ihyp-act Iv-act >>  Itv-indep > Ins (Fig. 5). Densities for Ihyp-act and Iv-act were in the range 60-100 pA/pF, whereas those for Itv-indep and Ins were much smaller at ~6 and 1 pA/pF, respectively. There were no statistical differences in the magnitude of the currents between WT and CF cells, at least for Iv-act, Itv-indep, and Ins. The mean density of the Ihyp-act current in the two genotypes was not markedly different (Fig. 5); however, this current was observed so infrequently in CF cells (see below) that a statistical comparison with WT was not possible.


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Fig. 5.   Densities of spontaneously active currents in nasal respiratory epithelial cells. Data from WT (open bars) and cystic fibrosis null (CF, filled bars) cells. For non-anion-selective current (Ins), n = 11 cells from 6 WT mice and 13 cells from 5 CF mice; for Itv-indep, n = 14 cells from 10 WT mice and 9 cells from 5 CF mice; for Iv-act, n = 12 cells from 9 WT mice and 6 cells from 4 CF mice; and for Ihyp-act, n = 13 cells from 9 WT mice and 1 cell from 1 CF mouse. Whole cell currents were measured at reversal potential of ±60 mV and normalized to input capacitance. Data were obtained 44 ± 3 s after start of the whole cell recording.

Frequency of spontaneously active currents in WT and CF cells. Figure 6 shows the frequency at which each of the four different spontaneously active currents were detected in WT and CF cells. These data were derived from I-V plots that were performed on average 44 ± 3 s after the onset of whole cell recording. Of the four identified currents, Itv-indep and Iv-act were observed at the same frequency in WT and CF genotypes (P > 0.05; chi 2). However, Ihyp-act was present at a significantly lower frequency in the CF cells (WT, 13/50 cells from 15 mice vs. CF, 1/29 cells from 6 mice; P = 0.01; chi 2) (Fig. 6). This is unlikely to result from a slow activation of Ihyp-act in CF cells following the onset of whole cell recording, because after 5 min of recording the frequency of detecting Ihyp-act remained significantly lower in the CF genotype (WT, 21/47 cells from 15 mice vs. CF, 3/23 cells from 3 mice; P = 0.01; chi 2). The reduced occurrence of Ihyp-act in CF cells was associated with an increased frequency of the Ins (WT, 11/50 cells from 15 mice vs. CF, 13/29 cells from 3 mice; P = 0.04; chi 2).


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Fig. 6.   Occurrence of spontaneously active currents in nasal respiratory epithelial cells. Bars represent percentage of cells displaying each type of current 44 ± 3 s after a whole cell recording was established. Data were from WT (open bars) and CF null (filled bars) cells. P values refer to difference between means for WT and CF cells. * Statistically significant difference. Data were from 50 cells from 15 WT mice and 29 cells from 6 CF mice.

Pharmacology of spontaneously active anion currents. We tested the following putative Cl- channel blockers on the three types of anion currents in WT and CF cells (n = 1-4 experiments with each blocker on each current): tamoxifen (10 µM), flufenamic acid (50 µM), glibenclamide (100 µM), DIDS (500 µM), and NPPB (10 µM). At the concentrations tested, none of these compounds had an inhibitory effect. However, Zn2+ and Gd3+ did block the currents.

Figure 7 shows that 500 µM ZnCl2 caused a voltage-dependent block of all three currents in WT cells, with preferential inhibition of the inward currents. Maximal inhibitory effects were observed ~1 min after addition of ZnCl2 to the bath solution, and the degree of inhibition of inward current was 57.9 ± 11.3% for Itv-indep (n = 5 cells from 3 mice, P = 0.03; WSRT), 83.2 ± 5.3% for Iv-act (n = 6 cells from 3 mice, P = 0.02; WSRT), and 64.5 ± 5.1% for Ihyp-act (n = 6 cells from 3 mice, P = 0.02; WSRT) (Fig. 7). There was no difference between the percent block of inward currents by ZnCl2 for Itv-indep and Ihyp-act, but Iv-act showed a significantly greater percent inhibition (P < 0.05; ANOVA + DMCT) (Fig. 7). These inhibitory effects of ZnCl2 were reversed after bath washout. In the case of Itv-indep and Ihyp-act, reversal was complete but the Iv-act current only partially recovered (Fig. 7). A similar blocking effect of ZnCl2 was observed on the spontaneously active Cl- currents in CF cells (n = 6 cells from 2 mice, data not shown).


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Fig. 7.   Inhibitory effect of ZnCl2 on spontaneously active Cl- currents in nasal respiratory epithelial cells. Currents were measured at reversal potential of ±60 mV and normalized to input capacitance. Open bars, control; hatched bars, 500 µM ZnCl2; filled bars, washout. Number of experiments on each current is shown in parentheses. A: Itv-indep; data were obtained 60 ± 12 s after addition of ZnCl2 to the bath and 51 ± 20 s after washout. B: Iv-act; data were obtained 69 ± 11 s after addition of ZnCl2 to the bath and 230 ± 76 s after washout. C: Ihyp-act; data were obtained 72 ± 12 s after addition of ZnCl2 to the bath and 115 ± 45 s after washout. Data were obtained from WT cells.

The inhibitory effect of 500 µM GdCl3 is shown in Fig. 8. Maximal block was observed ~1 min after addition of GdCl3 to the bath solution, and the degree of inhibition of inward current was 59.0 ± 10.7% for Itv-indep (n = 3 cells from 2 mice, P = 0.03; WSRT), 92.7 ± 5.1% for Iv-act (n = 4 cells from 2 mice, P = 0.0004; WSRT), and 94.5 ± 2.1% for Ihyp-act (n = 4 cells from 2 mice, P = 0.0001; WSRT) (Fig. 8). There was no difference between the percent block of inward currents by GdCl3 for Iv-act and Ihyp-act; however, GdCl3 was a less effective blocker of Itv-indep (P < 0.01; ANOVA + DMCT) (Fig. 8). These inhibitory effects of GdCl3 were not readily reversed on bath washout (Fig. 8).


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Fig. 8.   Inhibitory effect of GdCl3 on spontaneously active chloride currents in nasal respiratory epithelial cells. Currents were measured at reversal potential of ±60 mV and normalized to input capacitance. Open bars, control; hatched bars, 500 µM GdCl3; filled bars, washout. A: Itv-indep; data were obtained 62 ± 7 s after addition of GdCl3 to the bath and 177 ± 91 s after washout. B: Iv-act; data were obtained 31 ± 1 s after addition of GdCl3 to the bath and 210 ± 39 s after washout. C: Ihyp-act; data were obtained 30 ± 7 s after addition of GdCl3 to the bath and 225 ± 39 s after washout. Number of cells is shown below each data set. Data were obtained from WT cells.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

To date, all the patch-clamp studies that have been performed on the respiratory epithelium have utilized cultured cells (Refs. 11, 14 and see Ref. 3 for additional references). This means that 1) the cells may not be expressing a full channel complement, 2) channels cannot be localized to specific cell types, and 3) the membrane localization of the channels is uncertain if the cultured cells are not polarized and transporting. In this paper, we have described a new method for isolating viable ciliated respiratory cells from murine NE that are suitable for patch clamping. These cells are clearly polarized and retain the distinct morphological characteristic of respiratory cells in the native tissue: abundant cilia on their apical membrane. We took the fact that the cilia continued to beat and that beat frequency was increased by mechanical stimulation (19) as an indication that the cells were viable. Our isolated cells did not, however, appear to possess functioning gap junctions, a property that has previously been described for ciliated respiratory cells (19). To our knowledge, this is the first patch-clamp study to be performed on freshly isolated nasal respiratory cells.

Vm of WT Nasal Cells

The mean Vm of the isolated respiratory cells, measured in current-clamp experiments, was -31.7 mV. This is close to the Vm of -36 mV, measured using conventional microelectrodes, in freshly isolated human nasal respiratory cells (14). However, closer examination of the murine cells suggested that they could be classified into two groups. Type 1 cells, which made up 59.1% of the population, had an average Vm of -43.5 mV and a low conductance, whereas type 2 cells, which constituted 40.9% of the total, had a much lower Vm (-12.5 mV) and a high conductance.

Experiments in which we tested the effects of extracellular ion substitutions and of various channel blockers on Vm suggested that the membrane of type 1 cells was dominated by a K+ conductance, whereas type 2 cells possessed both K+ and Cl- conductances. The relatively high Cl- conductance in the type 2 cells would explain why these cells have a low Vm. Exposure of the in vitro murine NE to 100 µM amiloride caused a 37% inhibition of equivalent short-circuit current (10), and 54% of freshly isolated human nasal respiratory cells had a detectable Na+ conductance, as judged by a hyperpolarization in response to amiloride and the removal of extracellular Na+ (14). We could not detect an effect of amiloride alone on Vm in the isolated murine respiratory cells. However, small hyperpolarizations were observed if cells were exposed to amiloride after block of the K+ and Cl- conductances, suggesting that a small Na+ conductance is present.

Basal Cl- Conductances in WT and CF Nasal Cells

Previously, a basal Cl- conductance has been described in intact murine NE (10). A basal Cl- conductance has also been detected in cultured human NE and can be separated into a large apical conductance (which was absent in CF NE) and a smaller basolateral conductance that was unaffected in CF (25, 26). We have now extended this earlier work by establishing the biophysical and pharmacological characteristics of the Cl- conductance in unstimulated respiratory cells.

On the basis of their biophysical characteristics, three distinct basal Cl- conductances were identified in the nasal respiratory cells. All three conductances were detected at about the same frequency in WT cells; however, there were differences in their densities. Densities for Ihyp-act and Iv-act were in the range of 60-100 pA/pF, whereas that for Itv-indep was much smaller at ~6 pA/pF. This difference in current density may explain why the proportion of cells exhibiting a basal Cl- conductance in the whole cell experiments (75%) is substantially higher than the proportion of type 2 cells (40.9%) that we detected in the current-clamp experiments. If the low-density Itv-indep current is omitted, then the proportion of cells with a Cl- conductance falls to ~50%, which is much closer to the proportion of type 2 cells.

Whereas a panel of compounds (NPPB, DIDS, glibenclamide, tamoxifen, flufenamic acid) that have been shown to act as Cl- channel blockers in other cell types had no effect on the conductances in nasal respiratory cells, these conductances were blocked by ZnCl2 and GdCl3. At the dose we employed (500 µM), GdCl3 blocked all three conductances with equal efficacy. We were surprised by this blocking effect of Gd3+, since this trivalent cation is usually considered to be an inhibitor of stretch-activated cation channels (29). However, Gd3+ has recently been reported to block a volume-activated Cl- conductance in proximal tubule cells (18). ZnCl2 was a more effective blocker of Iv-act compared with Itv-indep and Ihyp-act. Previously, ZnCl2 has been reported to inhibit a hyperpolarization-activated Cl- conductance in rat superior cervical ganglion cells (5). Thus the pharmacological properties of the channels we have identified in the nasal respiratory epithelial cells are unusual, in that "traditional" Cl- channel blockers have no effect, whereas the channels are blocked by Zn2+ and Gd3+.

The simplest interpretation of our data is that 75% of WT nasal respiratory cells contain one of three types of Cl- channels that are open under unstimulated conditions. Although we cannot entirely exclude the possibility that Itv-indep (which had the lowest density) was also present in cells that exhibited Iv-act and Ihyp-act, our data from the CF cells argue against this. In the CF cells, the occurrence of Ihyp-act was markedly reduced and yet there was no increase in the frequency of either Itv-indep or Iv-act (only in Ins; see Fig. 6). Moreover, we have recently found that Ihyp-act can be completely and selectively inhibited by low-Cl- bath solutions and that no other Cl- currents are revealed under these conditions. Why individual ciliated cells should express only one type of basal Cl- conductance is unclear, but it could reflect either developmental or functional differences within the respiratory epithelium. The three Cl- conductances were observed at about the same frequency in WT cells. Therefore, it is probable that Iv-act and Ihyp-act make up the unstimulated cellular Cl- permeability of the airway epithelium, since they both have much higher densities than Itv-indep, and their voltage-dependencies suggest that they would be open at physiological Vm values.

Comparison With Other Cl- Conductances

The fact that the three Cl- conductances that we have identified were unaffected by NPPB, DIDS, glibenclamide, tamoxifen, and flufenamic acid indicates that they are not related to the CFTR and Ca2+-activated and volume-activated Cl- conductances that are well described in epithelial cells. Splice variants of CFTR have been reported in the NE (12). However, because we observed Itv-indep, Iv-act, and Ihyp-act in the CF nulls (albeit at a very much reduced frequency in the case of Ihyp-act) and none of these basal Cl- currents were affected by stimulants that increase intracellular cAMP (data not shown), it is unlikely that the nasal conductances could be a splice variant of the CF gene product.

Itv-indep. A conductance with similar time-independent kinetics to Itv-indep has previously been described in the basolateral membrane of gastric parietal cells where it is thought to play a "housekeeping" role, such as the regulation of Vm (13). However, unlike Itv-indep, the parietal cell Cl- conductance was rapidly blocked by NPPB with an inhibition constant of 12 µM (13).

Iv-act. To our knowledge, a conductance with similar properties to Iv-act, i.e., time-dependent activation at potentials greater than ±50 mV, has not been described previously. Iv-act was poorly selective for Cl-, having a Cl- permeability that was only 2.8-fold higher than that to NMDG+. Because NMDG+ is a large organic cation, the Cl- selectivity against Na+ is likely to be even lower. Thus, in a physiological situation, this channel may not select for anions over cations. Previously, a nonselective cation conductance with a relatively low selectivity for cations over anions (Na+-to-Cl- permeability ratio = 1.68), and which exhibits slight activation at depolarizing potentials, has been described in Caco-2 cells (16). This conductance is stimulated by protein kinase C and by cell shrinkage, effects that are thought to require participation of a regulatory protein that contains an ATP-binding Walker motif (16).

Ihyp-act. In terms of its biophysical and pharmacological characteristics, Ihyp-act bears a close resemblance to the ClC-2 Cl- channel that also activates at hyperpolarizing potentials and is insensitive to DIDS (23). ClC-2 is expressed in muscle, nerve, and epithelial tissues of the rat (23). A ClC-2-like conductance has also been identified in the granular duct cells of mouse mandibular glands (7). The salivary gland conductance is activated by hyperpolarizing potentials, inactivates at depolarizing potentials, and is unaffected by a variety of Cl- channel blockers, including NPPB, DIDS, and glibenclamide (7). A human ClC-2 analog (called ClC-2G or ClC-2alpha ) has recently been cloned from T84 cells (4) and has also been identified in both fetal and adult human lung (20). One particularly interesting aspect of our study was that Ihyp-act was present at a markedly reduced frequency in the CF nulls, indicating that this conductance may be modulated by CFTR in respiratory cells. CFTR is known to have an inhibitory effect on epithelial Na+ channels (22), and an outwardly rectifying Cl- channel in airway epithelium also requires CFTR for proper activation by agonists (8, 9).

Physiological Role of the Basal Cl- Conductances in Nasal Cells

Whole cell patch clamping cannot identify on which cell membrane a conductance is located. However, if Ihyp-act is being regulated by CFTR, then the conductance is most likely located on the apical plasma membrane. The location of the Itv-indep and Iv-act conductances remains speculative; however, they may contribute to the small basolateral Cl- conductance that has been detected in human NE cells and that is unaffected in CF (25, 26). It is difficult to predict the physiological function of the various Cl- conductances that we have identified without additional information about their membrane locations.

    ACKNOWLEDGEMENTS

We thank David Stephenson for skilled technical assistance.

    FOOTNOTES

This work was funded by the Biotechnology and Biological Sciences Research Council.

Present addresses: W. H. Colledge and R. Ratcliff, Physiological Laboratory, Univ. of Cambridge, Downing Street, Cambridge CB2 3EG, UK; R. Tarran, Dept. of Medicine, Univ. of North Carolina at Chapel Hill, 6017 Thurston-Bowles Bldg., Chapel Hill, NC 27599-7248.

Address for reprint requests: B. E. Argent, Dept. of Physiological Sciences, Univ. Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

Received 25 April 1997; accepted in final form 10 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Boucher, R. Human airway ion transport. Part two. Am. J. Respir. Crit. Care Med. 150: 581-593, 1994[Medline].

2.   Brezillon, S., F. Dupuit, J. Hinnrasky, V. Marchand, N. Kälin, B. Tümmler, and E. Puchelle. Decreased expression of the CFTR protein in remodeled human nasal epithelium from non-cystic fibrosis patients. Lab. Invest. 72: 191-200, 1995[Medline].

3.   Chan, H.-C., J. Goldstein, and D. J. Nelson. Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells. Am. J. Physiol. 262 (Cell Physiol. 31): C1273-C1283, 1992[Abstract/Free Full Text].

4.   Cid, L. P., C. Montrose-Rafizadeh, D. I. Smith, W. B. Guggino, and G. R. Cutting. Cloning of a putative human voltage-gated chloride channel (ClC-2) cDNA widely expressed in human tissues. Hum. Mol. Genet. 4: 407-413, 1995[Abstract].

5.   Clark, S., and A. Mathie. A hyperpolarization-activated chloride current in acutely isolated rat superior cervical ganglion (SCG) neurons (Abstract). J. Physiol. (Lond.) 485.P: 48P-49P, 1995.

6.   Cliff, W. H., and R. A. Frizzell. Separate Cl- conductances activated by cAMP and Ca2+ in Cl--secreting epithelial cells. Proc. Natl. Acad. Sci. USA 87: 4956-4960, 1990[Abstract].

7.   Dinudom, A., J. A. Young, and D. I. Cook. Na+ and Cl- conductances are controlled by cytosolic Cl- concentration in the intralobular duct cells of mouse mandibular glands. J. Membr. Biol. 135: 289-295, 1993[Medline].

8.   Egan, M., T. Flotte, S. Afione, R. Solow, P. L. Zeitlin, B. J. Carter, and W. B. Guggino. Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR. Nature 358: 581-584, 1992[Medline].

9.   Gabriel, S. E., L. L. Clarke, R. C. Boucher, and M. J. Stutts. CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. Nature 363: 263-266, 1993[Medline].

10.   Grubb, B. R., R. N. Vick, and R. C. Boucher. Hyperabsorption of Na+ and raised Ca2+-mediated Cl- secretion in nasal epithelia of CF mice. Am. J. Physiol. 266 (Cell Physiol. 35): C1478-C1483, 1994[Abstract/Free Full Text].

11.   Grygorczyk, R., and M. A. Bridges. Whole-cell conductances in brushed human nasal epithelial cells. Can. J. Physiol. Pharmacol. 70: 1134-1141, 1992[Medline].

12.   Hull, J., S. Shackleton, and A. Harris. Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells. Hum. Mol. Genet. 3: 1141-1146, 1994[Abstract].

13.   Kajita, H., S. Morishima, Y. Shirakata, T. Kotera, S. Ueda, M. Okuma, and Y. Okada. A mini Cl- channel sensitive to external pH in the basolateral membrane of guinea-pig parietal cells. J. Physiol. (Lond.) 488: 57-64, 1995[Abstract].

14.   Kunzelmann, K., S. Kathöfer, and R. Greger. Na+ and Cl- conductances in airway epithelial cells: increased Na+ conductances in cystic fibrosis. Pflügers Arch. 431: 1-9, 1995[Medline].

15.   Mery, S., E. A. Gross, D. R. Joyner, M. Godo, and K. T. Morgan. Nasal diagrams: a tool for recording the distribution of nasal lesions in rats and mice. Toxicol. Pathol. 22: 353-372, 1994[Medline].

16.   Nelson, D. J., X.-Y. Tien, W. Xie, T. A. Brasitus, M. A. Kaetzel, and J. R. Dedman. Shrinkage activates a nonselective conductance: involvement of a Walker-motive protein and PKC. Am. J. Physiol. 270 (Cell Physiol. 39): C179-C191, 1996[Abstract/Free Full Text].

17.   Ratcliff, R., M. J. Evans, A. W. Cuthbert, L. J. MacVinish, D. Foster, J. R. Anderson, and W. H. Colledge. Production of a severe cystic fibrosis mutation in mice by gene targeting. Nat. Genet. 4: 35-41, 1993[Medline].

18.   Robson, L., and M. Hunter. Role of cell volume and protein kinase C in regulation of a Cl- conductance in single proximal tubule cells of Rana temporia. J. Physiol. (Lond.) 480: 1-7, 1994[Abstract].

19.   Sanderson, M. J., A. C. Charles, and E. R. Dirksen. Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells. Cell Regul. 1: 585-596, 1990[Medline].

20.   Sherry, A. M., J. Cuppoletti, and D. H. Malinowska. ClC-2G, but not ClC-2, is present in adult and fetal human lung (Abstract). FASEB J. 10: 432, 1996.

21.   Smith, J., S. Travis, E. Greenberg, and M. Welsh. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996[Medline].

22.   Stutts, M., C. Canessa, J. Olsen, M. Hamrick, J. Cohn, B. Rossier, and R. Boucher. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[Medline].

23.   Thiemann, A., S. Grunder, M. Pusch, and T. J. Jentsch. A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356: 57-60, 1992[Medline].

24.   Wagner, J. A., A. L. Cozens, H. Schulman, D. C. Gruenert, L. Stryer, and P. Gardner. Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase. Nature 349: 793-796, 1991[Medline].

25.   Willumsen, N. J., C. W. Davis, and R. C. Boucher. Cellular Cl- transport in cultured cystic fibrosis airway epithelium. Am. J. Physiol. 256 (Cell Physiol. 25): C1045-C1053, 1989[Abstract/Free Full Text].

26.   Willumsen, N. J., C. W. Davis, and R. C. Boucher. Intracellular Cl- activity and cellular Cl- pathways in cultured human airway epithelium. Am. J. Physiol. 256 (Cell Physiol. 25): C1033-C1044, 1989[Abstract/Free Full Text].

27.   Winpenny, J. P., B. Verdon, H. L. McAlroy, W. H. Colledge, R. Ratcliff, M. J. Evans, M. A. Gray, and B. E. Argent. Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice. Pflügers Arch. 430: 26-33, 1995[Medline].

28.   Worrell, R. T., A. G. Butt, W. H. Cliff, and R. A. Frizzell. A volume-sensitive chloride conductance in human colonic cell line T84. Am. J. Physiol. 256 (Cell Physiol. 25): C1111-C1119, 1989[Abstract/Free Full Text].

29.   Yang, X.-C., and F. Sachs. Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243: 1068-1071, 1989[Medline].


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