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1 Department of Cell Biology and Anatomy
2 Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, Miami, FL, USA
3 Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina, Chapel Hill, NC, USA
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Abstract |
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(Received 9 July 2004;
accepted after revision 31 August 2004;
first published online 2 September 2004)
Corresponding author G. E. Conner: Department of Cell Biology and Anatomy and Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA. Email: gconner{at}miami.edu
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Introduction |
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Most secretions that contain SCN– also contain I–. I– is present at 10- to 20-fold higher concentrations in these secretions compared to serum (Brown-Grant, 1961), and I– is known to be transported across the thyroid epithelium in a Na+–I– symporter (NIS)-dependent fashion (De La Vieja et al. 2000). I– is used by thyroid peroxidase for the synthesis of thyroid hormones, but it can also be utilized by other haeme peroxidases such as lactoperoxidase. Mammary, salivary and gastric glands express NIS, and I– transport has been demonstrated in these tissues as well as in the kidney. NIS transcripts and protein have been shown to be present in the lungs (Wapnir et al. 2003), but there are no reports of functional NIS in the airway. SCN– is well known as a competitive inhibitor of I– transport by NIS, and NIS is known to transport SCN– in the thyroid gland (for review, De La Vieja et al. 2000). However, the function of NIS has not been experimentally linked with SCN– transport in these extrathyroidal tissues.
LPO enzymic activity has been shown to be a component of host defence in airways (Wijkstrom-Frei et al. 2003), and thus transport of SCN– across the airway epithelium might play a role in maintaining airway sterility. The regulation of SCN– secretion by epithelia provides a possible mechanism to manage peroxidase-mediated host defence activity on the luminal surface. For these reasons, we studied whether airway epithelia actively transport SCN–, and we characterized the properties of SCN– transport with regard to possible molecular components at both the basolateral (BL) and apical membranes.
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Methods |
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Unless otherwise stated all materials were obtained from Sigma Chemical Company (St Louis, MO, USA).
Cell culture
Human airways were from organ donors whose lungs were not to be used for transplant; they were obtained from the Life Alliance Organ Recovery Agency of the University of Miami. Local IRB-approved written consents for use of these tissues for research were obtained by the Life Alliance Organ Recovery Agency and conformed to the standards set by the Declaration of Helsinki. Airway epithelial cells were isolated, grown and re-differentiated at an air–liquid interface (ALI) on either 6.5 mm or 24 mm T-clear filters (Costar Corning, Corning, NY, USA) having a porosity of either 0.4 or 3 µm (only for permeabilization experiments) and coated with human placental collagen type IV as previously described (Bernacki et al. 1999; Nlend et al. 2002).
Transport experiments
All cultures had a resistivity of 
300 
cm. All experiments were performed under open-circuit conditions.
Time course of SCN– accumulation in the apical compartment. To determine the time course of SCN– accumulation from initial addition of [14C]SCN– to the BL medium to the point where [14C]SCN– concentration was near steady state, [14C]SCN– (85 µM, 50 mCi mmol–1, Amersham Pharmacia, Piscataway, NJ, USA) was added to the BL culture medium of fully re-differentiated ALI cultures, and Dulbecco's phosphate-buffered saline (PBS, pH 7.4, Gibco, NY, USA) was added to the apical surface of the culture. Unlabelled SCN– was not added to any of the media, thus the specific activity of [14C]SCN– was that supplied by the manufacturer. Samples of the apical PBS were collected at different times and transported SCN– was determined by liquid scintillation counting.
In experiments using cultures that were permeabilized basolaterally with 
-toxin from Staphylococcus aureus, ALI cultures were differentiated on 24 mm inserts with 3 µm pores. BL and apical compartments were washed with PBS and then 140 mM potassium gluconate, 0.33 mM CaCl2, 10 mM NaCl, 20 mM Hepes, pH 7.2, containing S. aureus
-toxin (10 000 U ml–1) was added to the BL compartment for 30 min at 37 °C in the absence of carbon dioxide. Permeabilization was apparent by the near absence of ciliary beating. The BL compartment was briefly rinsed with same buffer lacking -toxin, but containing 5 mM MgATP, 50 U ml–1 creatine phosphokinase, 10 mM creatine phosphate and [14C]SCN–. Addition of ATP and the ATP regeneration system to the BL compartment restored ciliary beating, suggesting the BL membrane was permeabilized as reported previously by others (Ostedgaard et al. 1992; Illek et al. 1999). After addition of [14C]SCN– to the BL compartment, appearance of [14C]SCN– at the apical surface was then measured by sampling small amounts of apical PBS followed by scintillation counting. Similar treatment of the apical surface did not decrease ciliary beating supporting the idea that -toxin was not able to permeabilize the apical membrane (Ostedgaard et al. 1992).
Unidirectional transport. To measure unidirectional transport of SCN– from the BL to apical compartments in the absence of accumulated apical SCN–, non-permeabilized cultures were washed with PBS and incubated in BL medium containing different concentrations of [14C]SCN– (50 mCi mmol–1) until the system approached steady state with regard to apical [14C]SCN– concentration, as determined from time course experiments described in the previous section. To measure I– transport, unlabelled I– was used. Transport of these anions was then determined by rapidly washing the apical surfaces of the cultures three times with PBS to remove anion accumulated on the apical surface. Following the third wash, additional aliquots (e.g. 500 µl per 24 mm filter) were placed on the apical surface for sequential 2 min incubations at 37 °C in humidified 5% CO2 until a steady flow of anions to the apical surface was obtained. Removal of washes after 2 min and replacement with fresh PBS prevent accumulation of anion in the apical compartment. BL medium was sampled after the last wash. [14C]SCN– in BL medium and apical washes was determined by liquid scintillation counting. All of the collected [14C]SCN– was soluble following 10% trichloroacetic acid (TCA) precipitation, showing that radiolabel was not covalently attached to protein.
A colourimetric I– assay (O'Kennedy et al. 1989) based on the Sandell–Kolthoff reaction was used to measure I– transport. A standard curve ranging from 0.01 to 0.6 µM KI was used. Reagent addition was precisely timed and transmittance at 414 nm was measured in a microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA) at 10 s intervals for 15 min after addition of ceric ammonium sulphate. The time points giving a linear range of transmittance with respect to standards (usually 5 min) were used to determine I– concentrations in samples.
To determine the transport of SCN– from the apical to BL compartment (back transport), apical surfaces of cultures were incubated with PBS containing [14C]SCN–, and BL medium was removed at 2 min intervals for liquid scintillation counting.
Apparent Km values were estimated by non-linear regression fitting to the Michaelis–Menten equation: V = VmaxS/(S + Km). Inhibition data were fit to V = VmaxS/{[(1 + i/Ki)S] + Km} – L, using the Km,app determined from the experiments described. L corrects for small amounts of non-inhibitable SCN– transport.
RT-PCR
RNA was extracted from normal human bronchial epithelial (NHBE)-ALI cultures using Trisolv (InVitrogen, NY, USA) and cDNA was obtained using SuperScript First Strand Synthesis System for RT/PCR kit (InVitrogen). NIS-specific oligonucleotide primers were designed according to Ajjan et al. (1998) (sense, 5'CTCCCTGTAACGACTCCAG3'; anti-sense 5'CTATCTCTATTACGGTGC3'). NIS cDNA was amplified by 35 cycles of 1 min at 94 °C, 55 °C and 72 °C, followed by a final 3 min elongation at 72 °C. PCR product was cloned using pGEM-T Easy Vector system (Promega, Madison, WI, USA) and sequenced using the ABI Prism 3100 Genetic Analyser (Applied Biosystems, CA, USA).
Immunolocalization
Tracheal tissue. Immunolocalization in tissue was performed according to Castro et al. (1999b) with slight modifications. Normal human tracheas were fixed in 4% paraformaldehyde and prepared for embedding in paraffin in a Microwave Tissue Processor (Microwave Materials Technologies Inc., Knoxville, TN, USA) according to the manufacturer's protocol using a non-xylene method. Embedding and sectioning were performed by the Histology Laboratory at the University of Miami Hospital and Clinics, Sylvester Comprehensive Cancer Center. Autofluorescence of de-paraffinized sections was reduced by incubating sections in sodium borohydride in PBS (5 mg ml–1). This was followed by quenching of endogenous peroxidase activity for 2 h in 3% H2O2 in methanol. Sections were subjected to antigen retrieval by incubating in 10 mM citrate buffer (pH 6) for 15 min at 80 °C followed by blocking for 1 h at room temperature in 5% normal goat serum (NGS, Chemicon, Tamecula, CA, USA) in PBS with 0.05% Tween-20 (PBS-T). Biotin autofluorescence was reduced utilizing Endogenous Biotin Blocking kit according to manufacturer's instructions (Molecular Probes, Eugene, OR, USA). Sections were incubated overnight at 4 °C with either anti-human NIS monoclonal antibody against amino acid residues 469–643 (Castro et al. 1999a), or non-immune mouse IgG (both from Chemicon, Tamecula, CA, USA) as a negative control. Both antibodies were used at 10 µg ml–1 in 1% NGS–PBS-T. Sections were incubated with affinity-purified goat anti-mouse IgG conjugated with horseradish peroxidase at a concentration of 0.02 µg ml–1 (KPL, Gaithersburg, MD, USA) for 45 min at room temperature followed by labelling using Tyramide Signal Amplification Kit (TSA, Molecular Probes) according to the manufacturer's protocol.
Cultures. ALI cultures were fixed in 4% paraformaldehyde and permeabilized with methanol for 10 min. Autofluorescence of fixed ALI cultures was reduced by incubating filters in sodium borohydride in PBS (5 mg ml–1). Cultures were subjected to antigen retrieval by incubating in 10 mM citrate buffer (pH 6) for 15 min at 80 °C followed by blocking for 1 h at room temperature in 100% NGS. Cultures were incubated overnight at 4 °C with either anti-human NIS monoclonal antibody or non-immune mouse IgG as a negative control; both were at a concentration of 10 µg ml–1 in 100% NGS. ALI cultures were incubated with purified goat anti-mouse IgG conjugated with AlexaFluor 555 (Molecular Probes) for 1 h at a concentration of 2 µg ml–1 in 100% NGS. Fluorescent images were obtained using a Zeiss LSM-510 confocal laser scanning microscope in the University of Miami Analytical Imaging Core Facility.
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Results |
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To assess the ability of human airway epithelial cells to accumulate SCN– in the apical compartment, de-differentiated human tracheobronchial epithelial cells were cultured in a two-chamber system that allowed re-differentiation at the air–liquid interface. Differentiation was evident by the presence of cells with beating cilia and goblet cells that secrete mucus. These cultures maintain a small volume of liquid on the apical surface (Matsui et al. 1998) and do not normally secrete LPO (as measured by enzyme activity and Northern blots, data not shown) that might metabolize transported SCN–, thus providing an ideal system to study the transport of this anion.
The accumulation of SCN– from the BL compartment to the apical compartment was first examined by adding [14C]SCN– to the BL medium and then following the time course of [14C]SCN– appearance at the apical surface. PBS, not containing SCN–, was added to the apical surface and then sampled at different times after addition of isotope and [14C]SCN– was determined by scintillation counting. After a brief lag, [14C]SCN– began accumulating at the apical side and to a 10.0 ± 1.1-fold higher concentration (mean ± S.E.M., n = 6) than that found in the BL medium. An example experiment is shown in Fig. 1A. Accumulation of SCN– on the apical side also occurred using culture media in both the apical (instead of PBS) and BL compartments. Addition of [14C]SCN– to the apical compartment did not result in accumulation in the BL compartment, and the appearance of apically applied [14C]SCN– in the BL compartment occurred at a much slower rate (see below).
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In identical experiments using separate cultures permeabilized with -toxin in the BL compartment, apical [SCN–] rapidly approached the BL [SCN–] but did not increase above that level (Fig. 1B). Permeabilization of the BL membrane was apparent by a dramatic decrease in ciliary beating that was restored upon addition of ATP and an ATP regeneration system in the BL compartment. Apically applied -toxin did not alter ciliary beating, consistent with other reports that -toxin does not permeabilize the apical surface. Thus, an intact BL membrane was apparently needed for concentration of SCN– at the apical surface of the ALI cultures.
The difference in accumulation of SCN– by the intact and permeabilized cultures is reflected in the rate at which SCN– appeared in the apical compartments (Fig. 1C). The rate of SCN– appearance in intact cells increased, presumably as the cellular compartment filled, and then decreased over several hours as the apical [14C]SCN– concentration increased and the system approached the steady state. In contrast, in the culture permeabilized with -toxin in the BL compartment, the rate of SCN– appearance reached a maximum after about 30 min and then declined to near zero for the remainder of the experiment. The 14C collected from the apical surface was soluble in 10% TCA indicating that the radiolabelled SCN– had not been bound to protein.
SCN– transport rates
To quantify transport rates, cultures were instead pre-incubated with [14C]SCN– in the BL medium until apical [14C]SCN– approached a steady value. The rate of appearance was then followed by adding and removing aliquots of PBS at the apical surface at 2 min intervals and determining the amount of 14C in the collected PBS as a measure of apical [SCN–]. These short washes insured that apical [SCN–] remained very low (< 1 µM) during transport measurements, thus transport was not inhibited by accumulation of SCN– in the apical compartment as suggested by Fig. 1.
The first wash showed a high level of isotope, suggesting that SCN– had accumulated in the small volume of apical surface liquid present during the pre-incubation period. Later washes contained a lower constant amount of isotope suggesting a constant rate of [14C]SCN– appearance (Fig. 2A). This constant rate obtained in the later washes increased with increasing BL [14C]SCN– concentration. Experiments were carried out on triplicate filters at each different BL [SCN–] used. The experiments used cultures from two different donors and 4 different days (n = 52 cultures). The rate was concentration dependent and appeared to be saturable at higher BL concentrations (Fig. 2B). Fitting the data to the Michaelis–Menten equation predicted an apparent Km of 69 ± 25 µM and an apparent Vmax of 24 ± 3 nmol h–1 cm–2 (fit coefficient ± S.D.) for transepithelial transport of SCN–. These experiments were conducted under open-circuit conditions, thus these constants are not corrected for any effects of electrical potentials. In addition, these values do not distinguish the different contributions of different subcellular compartments and surface membranes and thus are apparent values for the entire transepithelial transport process under the experimental conditions.
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Na+–I– symporter
Since SCN– can be actively transported by the NIS in thyroid gland and since NIS has also been detected in salivary gland, mammary gland and stomach (Ajjan et al. 1998; Spitzweg et al. 1998, 1999), all organs that transport SCN–, airway epithelia were probed for the presence of NIS mRNA and protein. RT-PCR using total RNA isolated from re-differentiated airway epithelial cultures showed a band of the expected size (Fig. 3). Sequence analysis of this band showed that it was 100% identical to NIS nucleotide sequence (GenBank accession no. U66088) suggesting that NIS is expressed in airway epithelia.
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NIS in thyroid gland concentrates I– in epithelial cells, and I– then exits via a channel in the apical membrane. To characterize the apical pathway by which SCN– exits airway epithelial cells, various inhibitors of anion channels were added to apical PBS washes to assess their ability to inhibit transport of SCN–. 4,4'-Dinitrostilbene-2,2'-disulphonic acid (DNDS, 100 µM), an inhibitor of anion exchangers and Cl– channels, had no effect on SCN– transport (Fig. 9), nor did 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 200 µM) (data not shown). In contrast, the sulphonylurea, glibenclamide (500 µM), an inhibitor of CFTR, blocked SCN– transport, as did the arylaminobenzoate, diphenylamine-2-carboxylic acid (DPC, 1 mM) (Fig. 9). Inhibition by glibenclamide and DPC was reversible on removal of the compounds, indicating that the cells remained viable during treatment (Fig. 9). 5-Nitro-2-(3-phenylpropylamino)-benzoate (NPPB) also reversibly inhibited SCN– transport (data not shown).
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Since CFTR-mediated Cl– current is increased in the presence of the ENaC blocker amiloride (Knowles et al. 1983), the effect of amiloride (100 µM) was also tested for its effect on SCN– transport. Inclusion of amiloride in apical washes was not associated with a significant increase in SCN– transport (Fig. 11), perhaps due to the smaller contribution of SCN– to the overall anion flux through CFTR.
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Discussion |
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The data suggested that SCN– transport occurred primarily via a transcellular route in ALI cultures and not a paracellular route. This was because SCN– was concentrated in the apical compartment in opposition to an electrical potential, transport was reversibly blocked by competitive inhibitors of NIS in the BL compartment and inhibitors of CFTR in the apical compartment, and required Na+ in the BL medium and was stimulated by treatments to increase intracellular cAMP. Thiocyanate accumulated to values similar to those reported previously for airway secretions (Wijkstrom-Frei et al. 2003), suggesting that the ALI cultures resemble in vivo epithelial SCN– transport. Importantly, these cultures do not make LPO under the conditions used for growth and differentiation. Finally, transported SCN– was not bound to protein by organification reactions.
Apparent kinetic constants estimated for SCN– and I– transport were within the range of published values for NIS in other tissues (Dohan et al. 2003). This was despite the fact that calculations of airway epithelial kinetic constants were performed under open-circuit conditions and did not take into account effects on transport by the apical membrane of the cells. Measurement of transport across intact epithelial layers under open-circuit conditions more closely resembles the physiological circumstances in airways, but in turn prevents accurate measurement of kinetic constants of the NIS in the BL membrane. Despite this, the measured Km,app for SCN– transport by airway epithelia (70 µM) was between that reported for SCN– transport by thyroid slices (30 µM, Wolff, 1964) and oocytes expressing recombinant NIS (96 µM, Eskandari et al. 1997). In thyroid samples, the Km values of NIS for SCN– and I– are identical, although in oocytes expressing NIS, Km values for these anions are threefold different (36 versus 96 µM). The Km,app and Ki,app for I– were dissimilar (111 versus 9 µM). Although the reasons for this are unclear, a similar difference was reported for recombinant NIS expressed in oocytes (Dohan et al. 2003). In this regard, both SCN– and I– have higher lipid-bilayer permeability than other anions of similar size that could contribute. Our experiments were conducted under open-circuit conditions and measured transport across the intact epithelium and thus are influenced by movement across the apical membrane as well.
The perchlorate Ki,app, using SCN– as a substrate, was comparable to that reported for NIS using I– as a substrate (Wolff, 1964; Eskandari et al. 1997) and thus the selectivity resembled that of NIS, perchlorate > I–
SCN–. These apparent kinetic constants, together with the Na+ requirement of transport, support the hypothesis that NIS may be the BL transporter responsible for uptake of SCN–. The absence of furosemide inhibition and the inhibition by perchlorate rule out a major contribution by the Na+–K+–2Cl– cotransporter.
Back transport studies showed low levels of apical to BL SCN– movement despite a transepithelial potential that favoured transport in this direction. Since apical to BL movement was not inhibited by perchlorate unless cells were first stimulated with forskolin and dibutyryl cAMP, this low level of back transport suggested that the paracellular route probably contributed a small amount to the overall movement of the anion in the BL to apical direction. Back transport was increased by stimulation of PKA and this increase was blocked by perchlorate in the BL medium, consistent with the idea that CFTR was involved in regulating movement of SCN– across the apical membrane.
In addition to the kinetic data, RT-PCR and immunolocalization support the presence of NIS in airway mucosa. Interestingly, immunolocalization suggested that NIS expression is heaviest in airway submucosal glands. Both RT-PCR and immunocytochemistry showed that ALI cultures used for these transport studies contained cells that expressed NIS basolaterally.
The observation that SCN– transport responded to apically applied compounds with an inhibition profile similar to CFTR, suggested that CFTR might regulate or possibly be the channel that passes SCN– to the apical surface. Like other Cl– channels, CFTR has been shown to carry SCN– although these studies primarily used SCN– as probe for channel properties (Tabcharani et al. 1993; Linsdell et al. 1997; Linsdell, 2001). The possibility that CFTR regulates SCN– transport in airway epithelia cells raises important questions regarding the effect of impaired CFTR activity on LPO-mediated airway host defence, and other potential effects of a loss of LPO activity on airway homeostasis in cystic fibrosis.
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). Mean values of the two filters are shown. In these intact cultures, after 240 min, the apical [SCN–] was tenfold higher than the BL [SCN–] despite an unfavourable electrical potential. B, the BL membranes of another culture were permeabilized with 
), and 306 (
) µM SCN– in the BL medium (concentrations determined from the specific radioactivity of BL medium). B, mean (±S.E.M.) basal transport rates from triplicate cultures at each BL [SCN–]. Cultures in B were obtained from two different lungs and assayed on 3 separate days, n
= 51 cultures. Fitting the data to the Michaelis–Menten equation by non-linear regression analysis gave apparent Km and Vmax for the transport process of 69 ± 25 µM and 24 ± 3 nmol h–1 cm–2, respectively (fit coefficient ±
S.D.) under open-circuit conditions.
X174.
