Evidence that Calu-3 human airway cells secrete bicarbonate

Michael C. Lee1, Christopher M. Penland1, Jonathan H. Widdicombe2,3, and Jeffrey J. Wine1

1 Cystic Fibrosis Research Laboratory, Stanford University, Stanford 94305-2130; 2 Cardiovascular Research Institute, University of California, San Francisco 94143; and 3 Children's Hospital Oakland Research Institute, Oakland, California 94609

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The Calu-3 cell line is being investigated as a model for human submucosal gland serous cells. In a previous investigation of basal short-circuit current (Isc) in Calu-3 cells, high levels of bumetanide-insensitive basal Isc (~60 µA/cm2) were measured in cells grown at an air interface. Basal Isc was reduced only 7% by bumetanide, and the largest component of basal Isc required both Cl- and HCO<SUP>−</SUP><SUB>3</SUB> in the bathing solutions. Because Isc could be partially inhibited by basolateral 4,4'-dinitrostilbene-2,2'-disulfonic acid and because the only known apical exit pathway for anions is the cystic fibrosis transmembrane conductance regulator, which has a relatively poor conductance for HCO<SUP>−</SUP><SUB>3</SUB>, it was concluded that most basal Isc is HCO<SUP>−</SUP><SUB>3</SUB>-dependent Cl- secretion [M. Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L690-L698, 1997]. We have now measured isotopic fluxes of 36Cl- and 22Na+ across short-circuited Calu-3 cells and found that virtually none of the basal Isc is Cl- secretion or Na+ absorption. Thus, in contrast to the earlier report, we conclude that the major component of basal Isc is HCO<SUP>−</SUP><SUB>3</SUB> secretion. Stimulation recruits primarily Cl- secretion, as previously proposed.

cystic fibrosis; Ussing chamber; epithelia; submucosal gland; cell culture

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CYSTIC FIBROSIS (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR), an anion channel located primarily in the apical membranes of epithelia. Lung disease is the most life-threatening consequence of CF, but it remains unclear exactly how defects in CFTR lead to lung infections. Submucosal gland serous cells are the predominant sites of CFTR expression in the human lung (4), so it seems important to understand the basis of ion transport in these cells. The Calu-3 cell line is being evaluated as a model for ion transport by human airway serous cells (5, 14, 17, 18).

An appreciable basal short-circuit current (Isc) is present in unstimulated Calu-3 cells (14, 17, 18). In previous work, the basal Isc was shown to be completely insensitive to apical amiloride (17, 18) and to be inhibited only slightly by bumetanide (18). Various blockers and ion-substitution experiments were used to identify three bumetanide-insensitive transport processes that account for most of the basal Isc. Phlorizin inhibits ~20% of the Isc by blocking Na+/glucose cotransport (18). Removing HCO<SUP>−</SUP><SUB>3</SUB> and CO2 from the bath eliminates most of the remaining Isc, whereas removing Cl- eliminates all phlorizin-insensitive basal Isc. Basal Isc was also reduced by acetazolamide, which slows the production of HCO<SUP>−</SUP><SUB>3</SUB> by inhibiting carbonic anhydrase, and by high levels of basolateral 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), which inhibits Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange (18).

Those results provided clear evidence for the importance of HCO<SUP>−</SUP><SUB>3</SUB> to basal Isc, but what role does HCO<SUP>−</SUP><SUB>3</SUB> play? Because virtually all Isc except that inhibited by phlorizin was lost when Cl- was removed from the medium (18), Cl--independent HCO<SUP>−</SUP><SUB>3</SUB> secretion (19, 21) was excluded.

The results of Singh et al. (18) are compatible with either HCO<SUP>−</SUP><SUB>3</SUB>-dependent Cl- secretion or Cl--dependent HCO<SUP>−</SUP><SUB>3</SUB> secretion. However, several findings led Singh et al. to favor HCO<SUP>−</SUP><SUB>3</SUB>-dependent Cl- secretion. First, apical Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange seemed absent because there was no inhibition of Isc with very high levels of apical DNDS or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Second, inhibition by basolateral DNDS indicated the apparent presence of a basolateral Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchanger. Third, CFTR has a relatively low conductance to HCO<SUP>−</SUP><SUB>3</SUB> (15). In the proposed model (18), basolateral Cl-/ HCO<SUP>−</SUP><SUB>3</SUB> exchange is used to concentrate intracellular Cl- concentration above the Cl- equilibrium potential (6, 13, 20, 22).

We have now tested this model by measuring transepithelial fluxes of 36Cl- and 22Na+. Contrary to the model, no net serosal-to-mucosal Cl- flux was detected during 15- to 30-min periods of basal Isc that averaged 46 ± 1 µA/cm2. In addition, no net Na+ absorption was detected. Because most basal Isc is eliminated by removal of either Cl- or HCO<SUP>−</SUP><SUB>3</SUB> from the medium, we conclude, in contrast to the earlier report (18), that the majority of basal Isc is Cl--dependent HCO<SUP>−</SUP><SUB>3</SUB> secretion.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The Calu-3 cell line was obtained and grown as in previous studies (14, 18). Cells were passaged with a 1:4 dilution or plated at 106 cells/cm2 onto human placental collagen-coated Costar Snapwell inserts (0.45-µm pore size, 1.13-cm2 surface area; Costar, Cambridge, MA). Cells grew on inserts at least 10 days before use. In contrast to previous studies, the filters were not cut from the inserts but were instead mounted in chambers that accommodated the inserts, thus avoiding edge damage. The medium was changed every 2-3 days with an air interface culturing in which medium was added only to the basolateral side of the inserts.

Commercially available Ussing chambers (World Precision Instruments, Sarasota, FL) were used to mount the Snapwell filters without edge damage. Both sides of the monolayers were bathed with 10 ml of bicarbonate-buffered (25 mM) Krebs-Henseleit solution (300 mosmol/l, pH 7.4 at 37°C when gassed with 95% O2-5% CO2). Drugs were added in small volumes from concentrated stock solutions. The transepithelial conductance was estimated at 2-s intervals by measuring responses to 1- or 2-mV amplitude pulses. Because oscillations in Isc were sometimes present, Isc was determined by measuring the area under the curve of Isc vs. time. Positive values represent anion secretion or Na+ absorption.

For flux measurements, 5 or 10 µCi of 36Cl- or 22Na+ were placed on either the mucosal or serosal side. One- to three-milliliter aliquots were removed from the cold side at 5- to 10-min intervals and replaced with fresh medium. Fifty-microliter aliquots were removed from the hot side at least every 30 min. To measure 36Cl-, three times the sample volume of scintillation fluid was added, and the mixture was counted in a Beckman LS6000 SC liquid scintillation counter. Samples containing 22Na+ were measured in a Packard 1500LS gamma counter. Net flux was determined as the difference between fluxes in the mucosal-to-serosal and serosal-to-mucosal directions.

All chemicals were reagent grade and, unless otherwise specified, obtained from Sigma (St. Louis, MO). Stock solutions of forskolin (Calbiochem, La Jolla, CA) in ethanol and thapsigargin in dimethyl sulfoxide were stored at -20°C. Stock solutions of bumetanide, phlorizin, and acetazolamide in dimethyl sulfoxide were stored at 4°C.

Statistics. Tissue pairs were matched within 0.5 mS/cm2 of baseline transepithelial conductance. Data are reported as means ± SE. Statistical significance was assessed with two-tailed Student's t-test. P values < 0.05 were regarded as statistically significant.

    RESULTS
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Methods
Results
Discussion
References

Measurement of 36Cl- fluxes during basal and stimulated Isc. In six matched pairs of tissues (mean transepithelial resistance = 232 ± 38 Omega  · cm2), isotopic Cl- fluxes were measured for periods of 15-30 min during which basal (unstimulated) Isc averaged 46 ± 1 µA/cm2. As shown in Fig. 1A and Table 1, no significant net flux of 36Cl- was observed during this period. Apical addition of 200 µM phlorizin decreased Isc by 12 ± 1 µA/cm2 but produced no significant change in Cl- flux. Bilateral application of 10 µM forskolin caused little change in either Isc or net Cl- flux. Subsequent addition of 300 nM thapsigargin increased Isc to 157 ± 20 µA/cm2 and net serosal-to-mucosal Cl- flux to 3.06 ± 0.47 µeq · cm-2 · h-1 (approx 82 ± 12 µA/cm2; n = 3 tissues). Thus, in each condition, a large portion of the Isc was not accounted for by Cl- secretion. This residual Isc, which may represent some combination of HCO<SUP>−</SUP><SUB>3</SUB> secretion and Na+ absorption, accounted for virtually all of the basal Isc and approximately one-third of the Isc stimulated by thapsigargin.


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Fig. 1.   Comparison of short-circuit current (Isc) and isotopic fluxes. A: Cl- fluxes. B: Na+ fluxes. Measured Isc, mean Isc for condition indicated; Cl- Isc Equivalent and Na+ Isc Equivalent, equivalent Isc calculated for 36Cl- and 22Na+, respectively; Residual Isc, difference between the two. Secretion of 36Cl- was only observed after treatment with thapsigargin (Tg), which in these experiments was always added after forskolin (Fsk).

                              
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Table 1.   Basal and stimulated Na+ and Cl- fluxes and corresponding Isc

Measurement of 22Na+ fluxes during basal and stimulated Isc. To determine the possible contribution of Na+ absorption to basal Isc, isotopic Na+ fluxes were measured in four matched pairs of tissues (mean transepithelial resistance = 408 ± 34 Omega  · cm2) for periods of 15-30 min during which basal (unstimulated) Isc averaged 42 ± 5 µA/cm2. The net serosal-to-mucosal flux of 22Na+ was 0.29 ± 0.14 µeq · cm-2 · h-1 (equivalent Isc of -8 ± 4 µA/cm2), which is not significantly different from zero and indicates that the basal Isc cannot be accounted for by Na+ absorption. (Basal Isc was lower than usual in this batch of filters, and phlorizin caused no change in Isc, suggesting that the Na+/glucose cotransporter was inoperative in these filters.) In two matched tissues, application of forskolin+thapsigargin caused Isc to increase to 104 µA/cm2, with no significant change in the flux of 22Na+ (Fig. 1B, Table 1).

    DISCUSSION
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Methods
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Past work (14, 17, 18) has shown that Calu-3 cells generate large basal and stimulated Isc values. The majority of stimulated Isc was shown to be Cl- secretion by flux measurements (17), but the ionic basis for basal Isc was not established. Singh et al. (18) proposed that most basal Isc was HCO<SUP>−</SUP><SUB>3</SUB>-dependent Cl- secretion, based primarily on the abolition of all basal Isc (except the phlorizin-sensitive component) after the removal of Cl- and the ability of basolateral but not apical DNDS to inhibit basal Isc. The present results negate that model and, instead, support a model in which most basal Isc is HCO<SUP>−</SUP><SUB>3</SUB> secretion, whereas most stimulated Isc is Cl- secretion (Fig. 2). This model is consistent with models for HCO<SUP>−</SUP><SUB>3</SUB> secretion in the duodenum and epididymus of rodents, (2, 8, 10), although in the latter tissues, an Na+/HCO<SUP>−</SUP><SUB>3</SUB> cotransporter may also play a role (2).


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Fig. 2.   Revised model of basal and stimulated Isc in Calu-3 cells. In this model, both Cl- and HCO<SUP>−</SUP><SUB>3</SUB> are secreted. At rest, Na+-K+-2Cl- cotransporter (NKCC) is inactive, cystic fibrosis transmembrane conductance regulator (CFTR) is partially active, and primarily NaHCO3 is secreted. When basolateral K+ channels are stimulated by elevated cytosolic free Ca2+, cell hyperpolarizes, NKCC is activated, and Cl- is secreted. Previous work showed that Calu-3 cells have a significant basal Isc that is insensitive to basolateral bumetanide and apical amiloride. Basal Isc is eliminated in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered or Cl--free solutions (18). Present results show that Cl- secretion or Na+ absorption cannot explain basal Isc, suggesting that HCO<SUP>−</SUP><SUB>3</SUB> secretion is occurring. Preliminary experiments indicate higher than usual activity of carbonic anhydrase in Calu-3 cells (J. Meezan and J. J. Wine, unpublished results), suggesting 1 way in which HCO<SUP>−</SUP><SUB>3</SUB> is accumulated for secretion. Exit pathway for HCO<SUP>−</SUP><SUB>3</SUB> is unknown. Possibilities include CFTR (2, 8, 10, 11, 15, 16), another apical transporter (indicated by ?) (2), or both. NHE, Na+/H+ exchanger.

The model shown in Fig. 2 is consistent with many, but not all, of our observations. It is consistent with HCO<SUP>−</SUP><SUB>3</SUB> and CO2 dependence of basal Isc, with an insensitivity of basal Isc to bumetanide, with a reduction in basal Isc by acetazolamide, and with a reduction in basal Isc by 5-(N-ethyl-N-isopropyl)-amiloride (Penland, unpublished observations). However, it does not account for the elimination of basal Isc in Cl--free medium. (It was this observation that originally suggested that basal Isc might be an HCO<SUP>−</SUP><SUB>3</SUB>-dependent Cl- secretion.) At present, we have no experimental data that can explain the Cl- dependence of basal Isc. However, if the model is correct as shown, then removal of Cl- must either open an equivalent basolateral path for HCO<SUP>−</SUP><SUB>3</SUB> exit, reduce the production of HCO<SUP>−</SUP><SUB>3</SUB> to a level that is in electrochemical equilibrium with external HCO<SUP>−</SUP><SUB>3</SUB>, or cause CFTR to close. [There is precedent for regulation of Cl--channel conductance by cytosolic anions in some cells (3).] The model also does not explain the inhibitory effect of DNDS on basal Isc. The model of Singh et al. (18) proposed that DNDS was inhibiting a basolateral anion exchanger, but such an exchanger is not compatible with the present model. A possibility consistent with the present model is that DNDS inhibits a basolateral Na+/HCO<SUP>−</SUP><SUB>3</SUB> cotransporter that elevates intracellular HCO<SUP>−</SUP><SUB>3</SUB> concentration.

Does HCO<SUP>−</SUP><SUB>3</SUB> exit the apical membrane exclusively via CFTR? Single-channel recordings (9) and measurements of Isc through basolaterally permeabilized Calu-3 monolayers (14) suggest that CFTR is the major and possibly only physiologically relevant anion channel in the apical membrane. Although HCO<SUP>−</SUP><SUB>3</SUB> conductance through CFTR has been controversial, a variety of measurements indicate that CFTR has a low but significant conductance to HCO<SUP>−</SUP><SUB>3</SUB> (7, 11, 16). In Calu-3 apical membranes specifically, Illek et al. (11) have shown that the average relative permselectivity for HCO<SUP>−</SUP><SUB>3</SUB> vs. Cl- is ~15%. Thus at least some HCO<SUP>−</SUP><SUB>3</SUB> could exit via CFTR, but the exact role of CFTR in HCO<SUP>−</SUP><SUB>3</SUB> secretion remains to be determined. Our measurements of Na+ fluxes, although sufficient to eliminate Na+ absorption as the basis for basal Isc, were not sufficient to rule out a contribution of Na+/HCO<SUP>−</SUP><SUB>3</SUB> cotransport (2) to basal Isc.

Speculation on functional consequences of CFTR mutations. In the human genetic disease CF, conductance through CFTR is lost. If the CFTR is the exclusive exit pathway for HCO<SUP>−</SUP><SUB>3</SUB> in Calu-3 cells and if Calu-3 cells mimic serous cells, loss of CFTR conductance will eliminate all basal and stimulated ion and fluid secretion, leaving only Na+/glucose absorption. Under these conditions, secreted macromolecules would be undiluted and trapped within the glands where unopposed absorption would desiccate them further. In addition, HCO<SUP>−</SUP><SUB>3</SUB> contributed by the glands would be absent from airway surface liquid, and the pH of the secreted fluid might be more acidic. An analogous condition has been simulated by pharmacologically blocking both Cl- transport and HCO<SUP>−</SUP><SUB>3</SUB> transport in bronchial submucosal glands from the pig, with the result that mucins accumulated in the glands (12).

Because of their abundant production of bactericidal compounds, serous cells are considered to be "the primary defensive cell of the mucosa" (1). Reduced fluid secretion by serous cells should reduce the dissemination of antibiotics in surface fluid and could contribute to the reduced mucosal defenses that are characteristic of CF. Therefore, it is of considerable interest to determine whether, in fact, all HCO<SUP>−</SUP><SUB>3</SUB> secretion in serous cells requires CFTR or whether alternate transporters exist in parallel with CFTR.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-42368 (to J. H. Widdicombe).

    FOOTNOTES

M. C. Lee was the recipient of a Howard Hughes Summer Research Fellowship from the Stanford University Department of Biological Sciences. C. M. Penland is a postdoctoral fellow of the Cystic Fibrosis Foundation.

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

Received 23 October 1997; accepted in final form 9 December 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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