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Am J Physiol Lung Cell Mol Physiol 273: L1208-L1219, 1997;
1040-0605/97 $5.00
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Vol. 273, Issue 6, L1208-L1219, December 1997

Calcium-stimulated Clminus secretion in Calu-3 human airway cells requires CFTR

Samina Moon, Meetpaul Singh, Mauri E. Krouse, and Jeffrey J. Wine

Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California 94305-2130

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl--dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl- channels. Typically, CF disrupts adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion but spares Ca2+-mediated secretion. However, in CF airway glands, Ca2+-mediated secretion is also greatly reduced. To determine the basis of Ca2+-mediated Cl- secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+ concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current (Isc) and transepithelial conductance (Gte) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (Delta ) in Isc and Gte, whereas forskolin stimulated variable and smaller increases. Delta Isc was decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid, and calixarene. Isc was measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl- conductance (GCl) that was decreased by DPAC but not by DIDS. Apical GCl could be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive GCl pathway in the apical membrane and display ~60% of maximum conductance at rest. Thus elevated [Ca2+]i increases K+ conductance to force Cl- through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.

cystic fibrosis; Ussing chamber; epithelia; submucosal gland; cell culture; amphotericin B; calcium ion; short-circuit current; chloride ion; cystic fibrosis transmembrane conductance regulator

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

SEROUS CELLS in human airways secrete antibiotic-rich fluid (2). Serous cells in submucosal glands of the human lung (9) and possibly in other regions of the distal airways (10) express high levels of cystic fibrosis transmembrane conductance regulator (CFTR). Primary cultures of submucosal gland serous cells also express CFTR and secrete Cl- in response to both cholinergic and adrenergic stimulation, with cholinergic stimulation being more potent (36). In these cultures, agents stimulate secretion to the extent that they elevate intracellular Ca2+ concentration ([Ca2+]i; see Ref. 37). For example, isoproterenol but not forskolin elevated [Ca2+]i, and isoproterenol but not forskolin was effective in stimulating short-circuit current (Isc; see Ref. 37). In cultured gland cells from cystic fibrosis (CF) subjects, responses to all mediators, including those that elevate [Ca2+]i, are greatly reduced (16, 34, 35). The diminution of Ca2+-mediated secretion in CF distinguishes submucosal gland cells from other organs such as sweat glands (22), and many organs in the mouse (8) in which Ca2+-mediated responses are altered little in the CF phenotype. However, it is consistent with the loss of Ca2+-mediated secretion in intestinal crypt cells of CF subjects (5).

Three general mechanisms might explain the reduction of Ca2+-mediated secretion in CF tissues (Fig. 1). 1) CFTR might be activated by a Ca2+-dependent mechanism (4, 29). 2) CFTR might be required to activate a different, Ca2+-dependent Cl- channel. 3) CFTR might be the predominant apical Cl- conductance (GCl) pathway and might also be substantially activated in unstimulated cells. If both conditions were met in the third mechanism, elevated [Ca2+]i could stimulate secretion by activating basolateral K+ channels and the bumetanide-sensitive Na+-K+-2Cl- cotransporter (14). Both of these effects would be eliminated if CFTR-mediated apical GCl were absent (23, 24).

The hypothesis to be tested in this paper is whether the bulk of cholinergically stimulated, Cl--mediated fluid secretion by human lung serous cells uses mechanism 3 in which CFTR channels, open at rest, serve as the exclusive conductance pathway for Cl- exit across the apical membrane and increased [Ca2+]i opens basolateral K+ channels to drive Cl- through the open CFTR channels.


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  		Fig. 1.   Three hypothetical mechanisms for cystic fibrosis transmembrane conductance regulator (CFTR)-dependent, Ca2+-stimulated Cl- secretion. A: CFTR is known to be influenced by protein kinase C (PKC; see Ref. 29). This effect could be a major activating pathway in some cell types. B: CFTR is known to influence other ion channels (27). An alternate Cl- channel in Calu-3 cells could require both Ca2+ and CFTR to function. C: If CFTR were normally open at rest, elevation of Ca2+ could produce secretion by opening basolateral K+ channels to provide a driving force for apical Cl- exit. [Ca2+]i, intracellular Ca2+ concentration.

To test this hypothesis, we used Calu-3 human airway cells, which have many similarities to submucosal gland serous cells (11, 15, 23). Calu-3 cells express a high level of CFTR, develop tight junctions, and secrete Cl- via apical CFTR channels. An analysis of their behavior using various pharmacological agents and nystatin-permeabilized cell sheets indicates that ~75% of the maximal apical GCl is already present in unstimulated cells and that the control of secretion is then driven by increases in cytosolic free Ca2+, which hyperpolarizes the basolateral membrane (23). However, because the agents used previously caused only transient increases in Isc (23), it remains possible that more sustained elevations in [Ca2+]i could activate CFTR (mechanism 1) or a different apical Cl- channel that is CFTR dependent (mechanism 2). Therefore, we have investigated the mechanism of Ca2+-mediated Cl- secretion in Calu-3 cells using thapsigargin to achieve sustained increases in [Ca2+]i without activation of other messengers that might actually truncate responses (17).

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The Calu-3 cell line was obtained frozen from the American Type Culture Collection (Rockville, MD). After being thawed, the cells were placed in tissue culture flasks (Costar, Pleasanton, CA) containing Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and penicillin-streptomycin and were grown at 37°C in an atmosphere of 5% CO2-95% air. Confluent monolayers were subcultured by trypsinization with a solution of phosphate-buffered saline (PBS), 0.04% EDTA wash, and 0.25% trypsin.

Cells were passaged in T75 flasks at a 1:4 dilution or plated at 106 cells/cm2 onto Costar Transwell inserts (0.45-µm pore size, 0.5-cm2 surface area; Costar, Cambridge, MA) coated with human placental collagen. After being plated, cells were maintained in culture at least 6 days before use, and medium was changed every 2-3 days.

We compared two kinds of culture conditions. In conventional ("submersed") culturing, medium was added to both sides of the filter. In air interface culturing, medium was added only to the basolateral side of the inserts. Air interface culturing markedly improves the differentiation of primary cultures of many kinds of epithelia (see Ref. 25 for further discussion).

Standard techniques were used in Ussing chamber studies. Filters on which cells had grown to confluency were cut from the plastic inserts and mounted between half-chambers so that they separated mucosal and serosal bathing solutions of identical ionic composition. Mean values for transepithelial conductance (Gte) in our experiments were 15 ± 4 mS/cm2 for air interface cultures (n = 11) and 7 ± 1 mS/cm2 (n = 25) for submersed cultures. In recent pilot experiments using confluent cells on intact human placental collagen-coated Snapwell filters, Gte was decreased to 6 mS/cm2 for air interface cultures (n = 11) and 2 ± 1 mS/cm2 for submersed cultures (n = 7). However, these monolayers also show reduced responses to mediators compared with what was observed in this series of experiments (C. Penland and M. Lee, unpublished observation). Both sides of the monolayers were bathed with 12 ml of Krebs-Henseleit solution, which contained (in mM) 128 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.2 glucose; osmolarity was adjusted to 320 mmol/l, and pH was 7.4 when gassed with 95% O2-5% CO2 at 37°C. Drugs were added in small volumes from concentrated stock solutions. Gas-lift oxygenators ensured proper oxygenation and stirring of the solutions. The transepithelial potential difference (Vte) was measured with agar bridges connected through calomel half-cells to a high-impedance electrometer. An external circuit used to bring the Vte to zero was connected to the backs of the half-chambers via agar bridges. The amount of electric current needed to maintain this voltage clamp was measured continuously on a chart recorder and on an LCIII computer using MacLab interface and software. Because the membrane was clamped to zero potential, it was regarded as short circuited, and the current that flowed under these conditions was called Isc. This Isc was considered to be representative of all the transport processes actively occurring across the tissue. Gte was estimated at 2-s intervals by measuring current changes in response to 1-mV pulses. Data are presented as means ± SE.

More direct measurements of apical membrane GCl were made by permeabilizing the basolateral membrane with amphotericin B (100 µM). This level of amphotericin B was determined as the concentration at which no response to bumetanide was seen (12). We then established an 11:1 serosal-to-mucosal ionic gradient for Cl- by bathing the basolateral surface in a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-based solution containing 140 mM Cl- and the apical surface in an identical solution except for the replacement of 91% of the Cl- with gluconate or aspartate.

All chemicals were reagent grade and, unless otherwise specified, were obtained from Sigma Chemical (St. Louis, MO). Stock solutions of forskolin (Calbiochem, La Jolla, CA) in ethanol, calixarene (a gift from R. Bridges and A. K. Singh) in water, and thapsigargin in dimethyl sulfoxide (DMSO) were stored at -20°C. Stock solutions of bumetanide in DMSO and ouabain in water were stored at 4°C. 4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS; obtained from Pfaltz & Bauer) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were dissolved in water. Fresh stocks were prepared daily for diphenylamine-2-carboxylate (DPAC) in DMSO. Fetal bovine serum was obtained from Hyclone.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Growth conditions had a marked effect on responses. Calu-3 cells grown at the air interface differ from submersed cultures in many ways. For example, air interface cultures have much larger basal Isc and lack the large, spontaneous drifts in Isc that are observed in many submersed cultures (25). The present experiments revealed the following general differences resulting from the two culture conditions.

Thapsigargin, which is membrane permeant, produced short-latency, fast-rising, multicomponent responses when applied to the apical side of monolayers grown as submersed cultures, but, to our surprise, it was ineffective on the basolateral side of submersed cultures. Thapsigargin was effective only when applied to the basolateral surfaces of air interface cultures, but the responses were long latency, slowly rising, and single component. The sidedness of thapsigargin stimulation was noted in the first study in which it was applied to native epithelia (6) but remains unexplained. Responses to forskolin also differed as a result of culture conditions. In submersed cultures, responses to forskolin were absent or small. For monolayers grown at the air interface, forskolin caused an average increase in Isc of ~40 µA/cm2, but this was still much smaller than responses to thapsigargin.

Despite these differences, we will propose a common model for Ca2+-stimulated Cl- secretion in cells grown in both kinds of conditions.

Agents that elevate [Ca2+]i produced large increases in Isc. Carbachol and histamine caused transient increases in Isc (Fig. 2 and Ref. 23). Basolateral application of carbachol caused responses that peaked within 5 s and fell to half-maximum within 16 s. The average peak responses to 10 or 100 µM carbachol were 17 and 77 µA/cm2, respectively, with a large amount of variability (Fig. 2C). For 100 µM carbachol, the average conductance increase at the peak of the response was 87 ± 38% (n = 9). Subsequent application of carbachol produced a response that was <10% of the initial response (n = 4). Basolateral application of 10 µM histamine caused an average peak increase of 7 µA/cm2 (n = 5), with a time course and variability similar to those observed after carbachol. Responses to carbachol and histamine were abolished by pretreatment with bumetanide (n = 3) or BaCl2 (n = 2), indicating that they are Cl- secretory responses that depend on both the bumetanide-sensitive Na+-K+-2Cl- cotransporter and Ba2+-sensitive basolateral K+ channels. Carbachol and histamine are known to elevate [Ca2+]i, but the intracellular messengers that they liberate can have additional effects (17). Therefore, we used thapsigargin to obtain elevated [Ca2+]i without additional effects of other intracellular messengers.


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  		Fig. 2.   Transient short-circuit current (Isc) responses to carbachol. Trace shows the largest Isc increase obtained with basolateral application of 100 µM carbachol. Vertical deflections on trace (see insets A and B) are responses to 1-mV pulses used to measure transepithelial conductance (Gte). Stimulation with 10 µM forskolin did not increase either Isc or Gte in this monolayer. Inset C: range of responses to carbachol observed in 9 monolayers. Filled circles are submersed cultures prestimulated with forskolin, filled squares are submersed cultures not prestimulated with forskolin, and open squares are air interface cultures not prestimulated with forskolin. In this and all following Isc traces, difference between baseline current and zero represents basal Isc. Properties of basal Isc were described in Ref. 25.

Thapsigargin selectively inhibits the Ca2+ pump of the endoplasmic reticulum to produce modest but sustained elevations in [Ca2+]i (31) and hence is useful for demonstrating a pure effect of [Ca2+]i on ion transport (7, 17). Thapsigargin caused large increases in Isc and Gte in almost all Calu-3 monolayers (Figs. 3 and 4). Figure 3 is an example of the short-latency (<10 s), biphasic responses that were typical for monolayers grown as submersed cultures. The early portion of the initial transient fell rapidly (time to half-amplitude ~3 min, n = 4). The second component was much more sustained, with a mean time to half-maximum of 37 ± 7 min (n = 13). The average peak changes in conductance after thapsigargin ranged from 52 to 117% in different conditions (Table 1).


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  		Fig. 3.   Isc responses to apical thapsigargin in submersed cultures. Each trace shows Isc across a monolayer of Calu-3 cells. Line thickness is caused by the voltage pulses used to measure conductance (see Fig. 2), which can not be resolved when prolonged recordings are compressed. A: stimulation with thapsigargin (300 nM, apical) caused a large increase in Isc and conductance and rendered the monolayer refractory to further stimulation with Ca2+-elevating agents. Stimulated Isc was only transiently affected by dimethyl sulfoxide (DMSO) vehicle (V) but was abolished by 200 µM bumetanide (Bm). Ba, barium. Marks near end of trace mark additions of compounds of no relevance. B: preaddition with bumetanide (10 and 100 mM) caused marked inhibition of response to thapsigargin. C: lack of response to forskolin (10 µM) and failure of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 200 mM) and calixarene (30 nM), both potent blockers of outwardly rectifying Cl- channels, to inhibit thapsigargin-stimulated Isc.


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  		Fig. 4.   Isc responses to basolateral thapsigargin and various inhibitors in monolayers grown at air interface. A: response to 300 nM thapsigargin applied basolaterally, followed by apical calixarene (30 nM), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; 3 mM), DIDS (400 µM; vertical lines), which had no effect, and diphenylamine-2-carboxylate (DPAC; 3.2 mM) added to both chambers, which abolished the response. B: response to 300 nM thapsigargin applied basolaterally, followed by 10 µM basolateral bumetanide. C: forskolin (10 µM, added at origin) produced a small increase in Isc that is not further increased by apical thapsigargin (300 nM, first vertical line), but basolateral thapsigargin caused a large response that was unaffected by apical application of calixarene, DNDS, and DIDS (vertical lines, same concentration as above). Isc was abolished by 3.2 mM DPAC added to both chambers.

                              
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  		Table 1.   Summary of response data

After thapsigargin, all other Ca2+-elevating agonists were ineffective. For example, Fig. 3A shows that a second addition of thapsigargin or two applications of carbachol evoked no further responses. Stimulated Isc was only transiently affected by DMSO vehicle (Fig. 3A).

The responses to thapsigargin were primarily mediated by basolateral Na+-K+-2Cl- cotransport because they were inhibited by bumetanide. When 10 µM bumetanide was added after thapsigargin, the change (Delta ) in Isc to thapsigargin was reduced by 69 ± 5% for submersed cultures (n = 10) and by 82 ± 5% for air interface cultures (n = 15; Fig. 3A). When bumetanide was added before thapsigargin, the response was reduced by 88 ± 8% for both types of cultures (n = 12). Bumetanide did not reduce Delta Gte. These results differ markedly from the small effect of bumetanide on basal Isc (25).

Response latencies (time to first rise) to basolateral thapsigargin in air interface cultures were ~30 times longer (~5 min) than to apical addition of thapsigargin in submersed cultures and lacked the initial peak seen in submersed cultures (compare Figs. 3 and 4). At least part of the markedly slower latency seen in air interface responses can be attributed to the lack of the early fast component of the response.

Oscillating responses to thapsigargin sometimes occurred in monolayers grown in either condition (Fig. 3). Such oscillations suggest that [Ca2+]i is oscillating with a similar rhythm in the majority of cells (13), but the mechanism whereby changes in [Ca2+]i are coordinated among the multiple cells (~106 cells) in the monolayer is unknown.

DIDS, usually considered a blocker of Cl- channels and exchangers (see below), in fact produced large, sustained increases in Isc when applied basolaterally to submersed cultures of Calu-3 cells, with a half-maximal effective concentration of ~120 µM (Fig. 5). (DIDS was not tested on the basolateral surface of air interface cultures.) Similar anomalous increases in Isc to basolateral DIDS were reported previously for T84 colonic tumor cells (7). In T84 cells, the responses were shown to result from increases in [Ca2+]i. The same mechanism is likely in Calu-3 cells because bumetanide inhibition of responses to DIDS was equivalent to its inhibition of thapsigargin responses (68%, n = 4) and because other Ca2+-elevating agents were relatively ineffective after basolateral DIDS. How DIDS elevates [Ca2+]i is not known (see Ref. 7 for discussion), but, like thapsigargin and unlike most agonists, DIDS causes sustained increases in Isc.


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  		Fig. 5.   Basolateral DIDS produces large increase in Isc. Apical application of carbachol (100 µM) caused a short-latency transient response, but little or no responses were produced by apical isoproterenol (Iso, 100 µM) or 10 and 100 µM DIDS. However, 200 µM DIDS applied basolaterally produced a large, short-latency response. Monolayer had been grown in submersed culture for 13 days before testing. Inset shows dose-response relation for basolateral DIDS. EC50, half-maximal effective concentration; Conc, concentration; Max, maximal.

We also attempted to elevate Ca2+ with ionomycin, but with escalating concentrations, we found that responses at threshold (0.2 µM) led to large, biphasic swings of Isc and irreversible, continuous increases in Gte within 5 min.

Elevation of intracellular adenosine 3',5'-cyclic monophosphate concentration usually produced small increases in Isc. Forskolin produced much smaller responses than did thapsigargin. For submersed cultures, forskolin failed to stimulate Isc in 9 of 14 preparations (e.g., Fig. 3C), and the mean Delta Isc for the 4 responding preparations was only 16 ± 6 µA/cm2. For air interface cultures, the mean Delta Isc to forskolin was 39 ± 12 µA/cm2 (n = 7), and there was a tendency for older cultures to develop larger responses to forskolin. Although this level of response would be considered robust in many preparations, it is only 23% of the Delta Isc to thapsigargin in these same preparations.

In 9 of 13 submersed cultures, forskolin also produced only small responses when applied after thapsigargin; the mean response to forskolin after thapsigargin in these nine monolayers was only 8 ± 3 µA/cm2. These small responses to forskolin are consistent with the hypothesis that basolateral K+ conductance (GK) is usually limiting for Isc in Calu-3 cells and that apical CFTR channels are normally open (hypothesis 3; see Ref. 23).

Exceptions to general results with thapsigargin and forskolin. We encountered several marked exceptions to the general findings that thapsigargin produces large increases in Isc and that forskolin produces small increases. In air interface cultures, the largest responses to forskolin approached the amplitude of responses to thapsigargin and oscillated with the same period as oscillations produced by thapsigargin (Fig. 6). In submersed cultures, exceptionally large responses to forskolin (Fig. 7) were observed after unusually small responses to thapsigargin in three experiments. The mean response to forskolin in these experiments was 147 ± 26 µA/cm2, a value ~20 times the mean for all forskolin responses and equivalent to the average response to thapsigargin. A model to explain both typical and exceptional cases is presented in the DISCUSSION.


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  		Fig. 6.   Atypical large responses to forskolin. Two of the largest responses to forskolin observed in air interface cultures are shown. A: change (Delta ) in Isc was abolished by 3.2 mM DPAC added to both sides. Compare these responses with the much smaller responses to forskolin or isoproterenol shown in Figs. 2-5. B: corresponding responses to thapsigargin from a paired filter. Time 0 was set at point of agonist addition; prior baseline was stable at the levels shown. Voltage deflections used to measure conductance were removed from these traces. Top trace in B is same data as in Fig. 4A but was rescaled for comparison with forskolin responses. ms572R, ms572L, ms573R, and ms573L indicate experiment numbers.


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  		Fig. 7.   Atypical small responses to thapsigargin and subsequent large responses to forskolin. In each of these submersed cultures, thapsigargin produced atypical small responses without a fast component. In A the response to thapsigargin was <= 20% of average; in B, the response was negligible. Subsequent application of forskolin produced exceptionally large, fast responses. This response pattern was observed in 3 of 13 experiments with submersed cultures and was never seen with air interface cultures.

Effects of inhibitors are consistent with CFTR-mediated, K+-limited Cl- secretion. CFTR is unusual among known epithelial Cl- channels with regard to its insensitivity to Cl- channel blockers. Swelling-activated (26) and some Ca2+-activated Cl- channels are blocked by stilbenes, but CFTR is not. Outwardly rectifying, depolarization-activated Cl- channels (ORDIC channels) that are often seen in excised patches are also blocked by DNDS (12, 26), DIDS (26), and calixarene. Thus these compounds can be used to determine if any of these channels contribute to Isc in Calu-3 cells. We applied DIDS (up to 600 µM), DNDS (up to 2 mM), and calixarene (30 nM) in various combinations to the apical surface of cells with no significant effect on basal Isc or Delta Isc stimulated by thapsigargin, forskolin, or their combination (Figs. 3-5 and Table 2).

                              
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  		Table 2.   Experiments with channel and transport blockers

In contrast to these negative results, responses to thapsigargin in both submersed and air interface cultures were reduced by basolateral applications of bumetanide, an inhibitor of basolateral Na+-K+-2Cl- cotransport (Figs. 3 and 4), by DPAC added to either side (Figs. 4 and 6), and by basolateral application of the K+ channel blockers BaCl2 or quinidine. Results with various inhibitors are quantified in Table 2 and Fig. 8.


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  		Fig. 8.   Inhibition of Delta Isc by channel blockers and transport inhibitors. Each bar shows mean %inhibition ± SE. Concentrations, no. of cultures, and exact numerical values for Isc and Gte are given in Table 2. All effects were stable except for BaCl2, which precipitated from the solution. For BaCl2, the %inhibition is the peak of the transient inhibition observed.

Spontaneous increases in Isc. All Calu-3 cultures, no matter how grown, had a significant basal Isc mediated mainly by bumetanide-insensitive, HCO<SUP>−</SUP><SUB>3</SUB>-dependent Cl- secretion, with a smaller component of electrogenic sodium-glucose transport (25). However, in addition to the basal Isc, ~40% of submersed cultures (vs. 0% of air interface cultures) spontaneously developed large increases in Isc within 30 min after being placed in the Ussing chamber. The properties of the spontaneous increases in Isc suggest that they represent Cl- secretion caused by increases in [Ca2+]i because they are inhibited by bumetanide (25) and because subsequent responses to thapsigargin are reduced by 88 ± 9% (n = 9).

Experiments on cell sheets with permeabilized basolateral membranes and transepithelial Cl- gradients. Results to this point show that agents that increase [Ca2+]i produce large increases in Isc and that forskolin is usually much less effective. Possible explanations for the efficacy of thapsigargin were outlined in Fig. 1. The small or absent Delta Isc in response to forskolin could arise if apical CFTR Cl- channels are constitutively active and if electrochemical equilibrium for Cl- across the apical membrane is not altered. In these cases, vectorial Cl- movement would be controlled indirectly by the basolateral exit pathway for K+.

To help decide among these possibilities, we determined the effect of different agonists on apical GCl by establishing Cl- or NaCl gradients across the monolayers and then permeabilizing the basolateral membrane with amphotericin B. Because of the frequent, spontaneous increases in Isc observed in submersed cultures (25), we only used the more stable air interface cultures for these experiments.

For basolaterally permeabilized monolayers ("apical membrane" preparations), with a 11:1 gradient of Cl-, addition of thapsigargin caused no change in Isc (Delta Isc = 0.8 ± 0.4 µA/cm2, n = 13) or Gte (Delta Gte = 0.2 ± 0.1 mS/cm2), whereas forskolin stimulated a significant Delta Isc of 33 ± 7 µA/cm2 and Delta Gte = 1.8 ± 0.6 mS/cm2 (n = 9; Fig. 9). These results extend previous results using different methods of permeabilization and transiently acting Ca2+ agonists (23) and reinforce the conclusion that the apical membrane of Calu-3 cells lacks Ca2+-activated Cl- channels.


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  		Fig. 9.   Apical membrane of Calu-3 cells lacks Ca2+-activated and stilbene-sensitive Cl- channels. Inset shows experimental arrangement: the monolayer of Calu-3 cells was placed in an 11:1 serosal-to-mucosal Cl- gradient, and ouabain and amphotericin B were applied to the basolateral membrane. Electrical recording of Isc was mainly generated by Cl- diffusion potential across apical membrane and paracellular pathway and Gte (trace thickness, see Fig. 2). Trace starts immediately after application of amphotericin B. Of agents applied, only forskolin and DPAC affected transapical Isc and Gte. Four vertical lines before DPAC mark additions of 30 nM calixarene, 400 µM DIDS, and 0.3 and 3 mM DNDS, respectively.

To assess the proportion of apical GCl that is active before stimulation, we established 11:1 gradients of Cl-, permeabilized the basolateral membrane, applied forskolin to maximize the apical GCl, and finally applied DPAC to eliminate apical GCl. The maximum Isc and Gte observed after forskolin was set to 100%, and the value after DPAC was set to 0%. The average proportion of Isc before application of forskolin was then measured and was determined to be 57 ± 5% (range 30-76%; n = 10; Fig. 10A). The average proportion of Gte before application of forskolin was 50 ± 10% (n = 6; Fig. 10B; see also Ref. 23).


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  		Fig. 10.   CFTR channels in apical membrane are active before stimulation. Using the same experimental arrangement, we determined the proportion of CFTR channels active before stimulation by setting the peak Isc and Gte after maximal forskolin to 100% and the minimum after maximal DPAC to 0%. A: example of a preparation with a low level of active CFTR channels at rest, measured via Isc. B: example of a preparation with a high level of CFTR activity at rest measured via Gte.

To determine the actual apical membrane GCl and the tight junction plus leak permeability, we assumed that DPAC eliminates apical GCl without affecting tight junction conductance and that the apical membrane has no appreciable conductance for cations. Given these assumptions, the difference in conductance between permeabilized monolayers before and after treatment with DPAC is a measure of apical membrane GCl, and the residual conductance is attributable to paracellular pathways, nonspecific leak pathways caused by edge damage, and incomplete inactivation of CFTR by DPAC. We obtained an average value of 8 mS/cm2 for apical GCl and 13 mS/cm2 for paracellular plus leak conductance (n = 24; Fig. 11).


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  		Fig. 11.   Dose-response curves for inhibition of Isc and Gte by DPAC. Curves have identical shapes with half-maximal effective concentration (500 µM) and a Hill slope of 1. This implies that the apical conductance is through a single type of Cl- channel.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

A model of Ca2+-stimulated Cl- secretion by Calu-3 cells. In the simple model outlined in Fig. 12, we hypothesize that Cl- secretion is controlled by just two switches, the apical Cl- (CFTR) and the basolateral K+ channel populations. The activity of these two channel populations vary independently and are controlled by intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration and [Ca2+]i, respectively. In Calu-3 cells grown at air interface, the apical CFTR conductance is ~60% of maximum at rest. The key features of the model are that 1) CFTR is the only apical Cl- channel and 2) a Ca2+-activated GK in the basolateral membrane is the key determinant of secretion. Basal secretion does not require Na+-K+-2Cl- cotransport, but stimulation recruits this transporter by an unknown mechanism (25). The major features of this model are consistent with previous models of Calu-3 cells (15, 23), with human submucosal gland cells (35, 37, 38), and with human tracheal epithelium (24).


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  		Fig. 12.   Model of Ca2+-stimulated Cl- secretion by Calu-3 cells. Basolateral membrane is at left. Key features of the model are that 1) CFTR is the only apical Cl- channel; 2) basolateral membrane contains a Ca2+-activated K+ channel but no cAMP-activated K+ conductance; 3) a substantial proportion of CFTR channels are active at rest; and 4) stimulation recruits Na+-K+-2Cl- cotransport by an unknown mechanism. Our experiments do not specify the mechanism(s) of increased apical Cl- conductance or basolateral K+ conductance, which could result from increased no. of channels (via vesicle insertion), increased open probability, or both.

Three findings support the hypothesis that CFTR is the only apical Cl- channel operating in Calu-3 cells. First, thapsigargin produced no change in apical GCl. Second, DIDS, DNDS, and calixarene caused no inhibition of apical GCl. Third, patch-clamp experiments from apical membranes of confluent Calu-3 cells show that CFTR channels accounted for virtually all Cl- channels observed (15). It remains possible that the apical membrane contains a non-CFTR Cl- channel, but, if so, the properties of that channel are considerably constrained by our results. Such a channel must be normally open, not sensitive to Ca2+, not inhibited by DIDS, DNDS, or calixarene, and not detectable with patch-clamp methods. It is also possible that a Ca2+-dependent channel exists but is either fully activated or fully inhibited by treatment of the basolateral membrane with amphotericin B.

We used DPAC to inhibit apical GCl. DPAC is often used to reduce or eliminate conductances mediated by CFTR, but DPAC is not a specific blocker of CFTR. Although it does cause an open channel block (25), it also severely reduces intracellular cAMP concentration levels by an unknown mechanism (18, 33). The inhibition of Isc and GCl that we observed probably represents a combination of channel block and reduction of intracellular cAMP concentration. This would not necessarily alter our conclusion, but the model is weakened by the possibility that DPAC is having still other unknown effects. However, sodium-glucose transport is not inhibited by DPAC, and Gte is decreased rather than increased; hence, monolayer integrity, the sodium-glucose transporter, and Na+-K+-ATPase activity would seem to be unaffected by high levels of DPAC.

If it is provisionally accepted that apical GCl is determined by CFTR, then evidence that CFTR channels are active in unstimulated preparations is based on the high levels of basal Isc seen here and in other studies (23, 25), the high apical GCl observed in unstimulated, basolaterally permeabilized preparations (Fig. 10, Table 1, and also Ref. 23), and the activity of single CFTR channels in unstimulated Calu-3 cells (15). We attempted to quantify the proportion of maximal (forskolin-stimulated) apical GCl that was open at rest by the methods outlined in Fig. 10. This method is accurate only to the extent that DPAC eliminates GCl. Because it is not certain that DPAC eliminates 100% of apical GCl, our estimate that 60% of maximal apical GCl is active at rest is a minimum estimate. Because DPAC eliminates all Cl- secretion in intact preparations (Table 2), we hypothesize that the remaining GCl arises from the paracellular and leak pathways. Our estimate that 60% of maximal GCl is active in basal conditions will be inaccurate to the extent that the basolateral membrane is not fully permeabilized by amphotericin B, the apical membrane is partially permeabilized by amphotericin B, or the paracellular pathway is affected by stimulation or by blockers.

Four observations support the hypothesis that basolateral GK is Ca2+ activated. 1) Thapsigargin, which elevates [Ca2+]i without elevating other intracellular messengers, is the most potent stimulus we have found for stimulating Isc. 2) Thapsigargin had no effect on apical GCl but did markedly increase Gte. 3) Basolateral Ba2+ inhibited the thapsigargin stimulation of Isc. 4) Forskolin often had little or no effect on secretion, suggesting that a cAMP-activated GK is either absent or is a minor, fully active component of GK in Calu-3 cells.

Although simple, the model can account for both the typical and atypical responses that we observed. The modal response for Calu-3 cells was a small response to forskolin and a large response to thapsigargin, with an average response to the combined agents that was only slightly larger than to thapsigargin alone. These results indicate that cAMP doesn't increase basolateral GK in Calu-3 cells because if it did forskolin should always produce large responses. More than 90% of our preparations fit the above description, but, as shown in Table 3, we also encountered monolayers that behaved as if both channels were closed (initially refractory to either agent), as if both channels were open (spontaneous increases in Isc seen only in submersed preparations), and as if K+ channels were active but CFTR was relatively inactive (small response to thapsigargin but large response to forskolin).

                              
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  		Table 3.   Four possible configurations of apical and basolateral channels and predicted consequences or responses to elevations of [Ca2+]i and [cAMP]i

Comparison of models of Cl- secretion by Calu-3 cells and T84 cells. The proposed mechanism for Cl- secretion in Calu-3 cells shares features with T84 cells, which are a model for colonic crypt cells. In T84 cells, most evidence is consistent with the hypothesis that confluent sheets of polarized cells have CFTR as the primary or exclusive apical GCl (30), and many Isc experiments were interpreted to mean that Ca2+-mediated secretion resulted from activation of Ca2+-dependent, basolateral K+ channels (7, 17), although in at least one experiment an additional DIDS-sensitive conductance was observed (20). A major difference between T84 and Calu-3 cells is the extent to which CFTR channels are open in Calu-3 cells. A possible difference is that T84 cells may have a significant cAMP-activated basolateral GK (28), whereas Calu-3 cells do not. Finally, unlike Calu-3 cells, T84 cell Isc is entirely inhibited by bumetanide.

How well do Calu-3 cells model human submucosal gland serous cells? Human gland serous cells are relatively inaccessible, and the study of Calu-3 cells is just beginning, so comparisons are necessarily limited. In the only single-channel patch-clamp study of human submucosal gland serous cells, channels having the properties of CFTR channels were observed, but Ca2+-dependent Cl- channels were not (3). In Ussing chamber comparisons of gland cells from normal and CF subjects, it was found that Cl- secretion in CF cells was greatly reduced, not only to agents that elevated intracellular cAMP concentration but also to agents that elevated [Ca2+]i, consistent with the proposed model (35).

Several recent reports might be taken as evidence against the model and in favor of Ca2+-dependent Cl- channels in human submucosal gland serous cells. Yamaya et al. (38) found that Cl- secretion by human gland cells could be stimulated by purinergic agents and the responses inhibited by DIDS. Although DIDS inhibition might be considered suggestive of a non-CFTR Cl- channel, it seems more likely that DIDS was acting as an antagonist of the P2Y receptor (19). A series of papers using feline submucosal gland serous cells has presented evidence for Ca2+-dependent Cl- channels in that species (e.g., Ref. 21). Although human preparations were also studied sometimes, all records presented are from cats. In summary, we know of no compelling evidence for Ca2+-dependent Cl- channels in human submucosal gland cells. Thus Calu-3 cells and human submucosal gland serous cells may be similar both biochemically (11) and electrophysiologically, but much additional work on both types of cells is necessary.

Implications for CF. We hypothesize that Calu-3 cells are a model for submucosal gland serous cells (11, 15, 23). If true, it follows that CF serous cells will fail to secrete to cholinergic agents (35) because such secretion depends on apical CFTR channels that are open at rest (15, 23, 25). Our present understanding of submucosal gland function is that CFTR expression is high in serous cells and low or absent in mucous cells (9). Hence, in CF, the hypothesis is that submucosal glands will secrete mucus that is not hydrated by antimicrobial-rich fluid from serous cells. A similar imbalance between fluid and mucus secretion may play out in the small airways where serous and mucous cells are located at the surface. The evaluation of this hypothesis, and other hypotheses about salt composition of airway fluid, will require new methods and perhaps new model systems.

It is often stated that cAMP-mediated secretion is defective in CF but that Ca2+-mediated secretion is intact. This claim is the basis for therapies designed to circumvent CF symptoms by activating latent or underutilized Ca2+-activated Cl- channels (alternate Cl- channels) in the lungs and other organs affected in CF. The generalization is reinforced by evidence that CFTR Cl- channel activity requires phosphorylation by cAMP-dependent protein kinase, with less important roles being played by Ca2+-activated kinases. However, in human colonic epithelium, CF causes a loss of Cl- secretion that is stimulated by both Ca2+ and cAMP-mediated pathways (5, 32). Human lung submucosal gland cells also show a severe reduction in Ca2+-mediated secretion in CF (16, 35).

Calu-3 cells are not a good model for most airway surface epithelial cells, which, at least in the upper airways, contain low levels of CFTR but higher levels of epithelial Na+ channel. Surface cells of the upper airways are primarily involved in absorption of Na+, Cl-, and fluid (34), whereas submucosal gland serous cells are primarily involved in fluid secretion. Airway surface cells can be stimulated to secrete, and, unlike Calu-3 cells, surface cells contain Ca2+-activated Cl- channels (1, 8).

An emerging theme in CF research is that, in both mice and humans, organs that contain alternative GCl do not develop CF disease (8), suggesting either that GCl is the critical function that CFTR performs or that alternative Cl- channels can also mimic other functions of the CFTR. But this creates an enigma: if human airway surface epithelia contain an abundant, alternate GCl, why are the lungs subject to CF disease? Our hypothesis avoids that enigma: the loss of apical GCl in submucosal gland serous cells is critical to human CF lung disease because these cells lack alternate Cl- channels. The predicted consequence is a reduction in secretions of antibiotic-rich fluids that are thought to be a crucial component of airway mucosal defenses (2, 11, 15, 23, 25, 35).

The implication of this hypothesis is discouraging because it suggests that the strategy of activating "bypass" Cl- channels may not work for submucosal gland serous cells. However, it remains possible that activation of alternate Cl- channels in surface epithelia might prevent or at least slow the course of CF lung disease.

    ACKNOWLEDGEMENTS

We thank Tina Law, Ilynn Nepomuceno, and Clare Robinson for cell culture, R. Bridges and A. K. Singh for a gift of calixarene, and Chris Penland for suggestions on the manuscript.

    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-42368 and DK-51817, Cystic Fibrosis Research, Inc., Cystic Fibrosis Foundation, Ron and Kay Presnell, and Patricia Bresee. S. Moon and M. Singh were recipients of a Cystic Fibrosis Foundation Student Traineeship and a Howard Hughes Summer Research Fellowship.

Address for reprint requests: J. J. Wine, Cystic Fibrosis Research Laboratory, Bldg. 420 (Jordan Hall), Stanford University, Stanford, CA 94305-2130.

Received 11 February 1997; accepted in final form 21 August 1997.

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Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(6):L1208-L1219


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